우리는 기능 유전체학 시설과 분광법 및 Bioimaging 시설, NIBB 핵심 연구 시설 및 모델 식물 연구 시설, NIBB 생물 자원 센터의 직원을 감사드립니다.

식물 씨앗에서 총 RNA 1. 추출 <ol><li> 버퍼 세트, 스핀 열, 1.5 및 2.0 mL의 폴리 프로필렌 튜브 및 뉴 클레아없는 1.5 ML의 폴리 프로필렌 튜브를 준비합니다. </li><li> (V / W) (이하, PVP라고 함), 분자 생물학 등급의 폴리 비닐 피 롤리 돈을 적극적 RNA 추출 및 와류에 대한 용해 완충액 셀 1 % 추가. 완전히 용해시키기 위해 25 ℃에서 20 분 동안 인큐베이션. 20 분 배양 후, 기포의 형성을 방지하기 위해 거꾸로 튜브를 부드럽게 돌려서 버퍼를 섞는다. 사용하기 전에 실내 온도 (15-25 °에 C)에 보관하십시오. </li><li> 얼음에 1.5 mL의 폴리 프로필렌 튜브에 애기 장대 식물과 장소에서 수확 과일입니다. 알루미늄 판에 장소 과일 4 ° C로 유지하고 스테레오 현미경 열매에서 씨앗을 격리 할 것. <ol><li> 액 N의 튜브를 배치 즉시 얼음에 알루미늄 랙에 저장되어있는 1.5ml의 폴리 프로필렌 튜브로 절연 종자 (종자 약 200)를 놓고itrogen. </li></ol></li><li> 액체 질소에서 튜브를 제거하고 얼음에 알루미늄 랙에 반환. 4 ° C에서 1,000 XG에서 1 % 1 분 동안 PVP와 원심 분리기를 포함하는 버퍼 100 μL를 추가합니다. </li><li> 얼음 알루미늄 랙 튜브를 유지하면서 60 초 동안 모터 분쇄기를 사용하여 스테인리스 봉과 시료 균질화. </li><li> 1 % PVP를 함유하는 완충액 550 μL를 추가 거꾸로 튜브를 돌려 버퍼를 부드럽게 혼합하고 25 ° C에서 10 분 동안 배양한다. </li><li> 5 25 ° C에서 8,000 XG에 분하고 새로운 1.5 ML의 폴리 프로필렌 튜브로 전송 상층 액 550 μL에 대한 원심 분리기. </li><li> 5 25 ° C에서 10,000 XG에 분하고 새로운 1.5 ML의 폴리 프로필렌 튜브로 전송 상층 액 450 μL에 대한 원심 분리기. </li><li> RNA 추출을위한 세포 용 해물로 뜨는을 사용합니다. 이하, 시판 키트 제조업체의 지침에 설명 된 절차를 따르십시오. </li><li> 용출RNA는 낮은 수준의 전 사체의 발현 양상을 모니터링하기 위해 RNA의 충분히 높은 농도를 확인하기 위해 용출 버퍼의 최소량을 사용. 사용까지 깊은 냉동고에서 RNA를 저장합니다. </li></ol> RNA 품질의 2. 확인 <ol><li> 총 RNA를 녹여 부드러운 도청에 의해 혼합하고 얼음에 알루미늄 랙에 튜브를 배치합니다. </li><li> microvolume 분광 광도계를 사용하여 RNA 농도 A260 / A280 비율 및 A260 / A230의 비율을 측정한다. </li><li> 70 NG / μL 최종 농도의 RNase가없는 물에서 총 RNA를 희석한다. </li></ol> 씨앗에서 총 RNA의 3 역전사 <ol><li> 상업 키트에서 총 RNA와 버퍼 세트를 해동. 부드러운 태핑 후 얼음에 키트에서 효소를 유지합니다. 클레아없는 0.2 ML의 폴리 프로필렌 튜브를 준비합니다. </li><li> 0.2 ML의 polypropy 5 총 RNA의 μL, 1 μL 올리고의 DT 프라이머 (50 μM), 1 임의의 μM 6 량체 (50 μM)를 추가얼음 렌 튜브. </li><li> 37 ºC에서 15 분 동안 품어과 얼음에 배치합니다. </li><li> 이하, 역전사-PCR 키트 제조업체의 지침에 설명 된 절차를 따르십시오. </li><li> 사용하기 전에 얼음에 역전사 제품을 놓습니다. </li></ol> 4. 정량적 실시간 PCR 분석 <ol><li> 제조자의 프로토콜을 사용하여 표적 유전자 서열을 보유하는 플라스미드를 구축. </li><li> 표준 곡선에 대한 DNA 템플릿 100-500,000 복사 / μL로 농도를 조절합니다. </li><li> 증류수로 다음의 cDNA 솔루션 (100 일)을 희석. </li><li> 이 희석 된 cDNA 솔루션의 μL 및 정량적 실시간 PCR 키트에서 마스터 믹스로 표준 곡선의 플라스미드를 추가합니다. </li><li> 30 초 동안 95 ° C 다음 5 초 동안 95 ° C에서 40주기와 35 초 동안 60 ° C : 다음 사이클링 조건을 사용하여 실시간 PCR을 설정합니다. </li><li> 사람에 따라 복사 번호 분석실시간 PCR 시스템 제조 회사의 지시. </li></ol>

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The authors have nothing to disclose.

유전자 발현 프로파일은 식물 생리에 대한 우리의 이해에 기여; 따라서, 특정 RNA 분리 방법은 각각의 샘플 조건 9-12 개발되었다. 우리는 씨앗에서 RNA 분리하는 동안 억제와 RNA는 실리카 막에 결합하는 것은 심각하게 저해 것을 발견 과정을 조사 하였다. 오일, 단백질, 폴리 페놀 다량의 RNA 분리를 억제한다. 우리는 RNA 실리카 막에 결합하는 과정 전에 분해 용액으로 이들 화합물을 제거하는 RNA 추출 과정을 수정. 중요한 변형 (1.7 단계 1.8) 1 % PVP (단계 1.2) 원심 분해 용액의 상청액 수집의 첨가를 포함했다. 1 % PVP의 추가의 목적은 폴리 페놀을 제거하는 것이다. 우리가 사용하는 PVP의 효과적인 농도를 조사하고, 1 % PVP이 프로세스에 가장 효과적인 농도 인 것을 확인했다. 원심 분리를 용해 액 상등액을 수집 목적오일 및 단백질을 제거하는 것이다. 상등액을 상등액 오일 층과 튜브의 펠렛을 유지 않도록주의를 수집한다. 이러한 두 가지 간단한 변형 대폭 중간에 씨앗 개발 후기 단계 (표 1)로부터 복구, A260 / A280 비율 및 절연 RNA의 A260 / A230 비를 개선 하였다. 표준 프로토콜의 간단한 수정을 대표하는 우리의 방법은, RNA 분리에 대한 시판 키트를 사용합니다. 따라서, 우리의 방법은 염화 리튬을 사용하는 페놀 - 클로로포름 추출 및 에탄올 침전 등의 번거로운 추가 단계를 필요로하지 않고 고순도 종자로부터 RNA를 분리하는 실용적이다. 우리의 방법은 수정이 석유와 단백질이 풍부한 작물 종자 잘 작동을 나타내는, 또한 성숙한 유채와 콩 씨앗 (표 2)와 함께 사용하기에 효과적이다. 4 DAF 씨앗 (표 1)과 젊은 루트에서 RNA의 회수율의, 잎과 줄기는 우리의 방법은 기름과 단백질 가난한 조직에서 RNA 분리에 적합하지 않은 것을 나타내는, 우리의 방법을 사용 낮았다. 따라서, 방법은 그러한 종자 및 과일과 같은 오일, 단백질, 폴리 페놀을 함유하는 조직으로부터 RNA의 분리에 유리하다. 전반적으로, 우리의 방법은 RNA를 정제 페놀, 클로로포름, 또는 추가의 공정을 사용하지 않고 식물의 씨앗을 고도로 정제 된 RNA를 획득하기에 적합하다. 이 방법은 종자의 유전자 발현의 더 정확한 프로파일을 용이하게한다.

식물은 다음 세대를 야기 종자를 생산한다. 씨앗은 후 germinative 성장을위한 오일, 탄수화물, 단백질, 같은 저장 매장량의 많은 양의 축적. 인간은 식품 및 동물 사료의 소스로 종자 저장 보유고를 활용, 따라서 식물 씨앗은 전 세계적으로 식용 유기 물질의 주요 공급 업체 중 하나입니다. 종자 생산량 증가는 식물 과학에서 중요한 도전이다. 종자 저장 매장량 식품 및 산업용 소재의 상업적 가치있는 소스이기 때문에, 이러한 매장량의 신진 대사의 규제를 기본 분자 메커니즘 널리 1-6 조사되었다. 또한 이러한 메커니즘을 해명하는 것은 작물 종자 수확량을 증가 도움이 될 것입니다. 씨앗은 수정 후 식물의 난소에서 개발, 그들은 발달 단계 1,6,7의 시리즈를 통해 성숙. 또한 분자 메커니즘 기본 종자 개발을 이해하는 상세한 필요씨앗, 현상하는 일련의 정확한 유전자 발현 프로파일을 생성한다. 그러나, 식물의 씨앗 오일, 단백질, 탄수화물, 폴리 페놀의 다량 어려운 유전자 발현의 정확한 프로파일을 배제 고순도 RNA를 분리 할 수 ​​있습니다. 여기서는 오일, 단백질, 폴리 페놀을 다량 함유 종자로부터 RNA를 분리하기위한 효율적인 방법을 소개한다. 이 방법을 사용하여, 연구자는 고도로 정제 된 RNA를 제조 할 수있을 것이다. 이러한 RNA 개발 및 성숙 종자에서 종자 저장 매장량의 대사 조절을 제어하는 ​​핵심 유전자의 전사 변화를 모니터링하는 데 유용 할 것입니다.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RNeasy Plant Mini Kit</td>
			<td style="width:81px;">QIAGEN</td>
			<td style="width:136px;">74904</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">polyvinylpyrrolidone</td>
			<td style="width:81px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5288-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HOMOGENIZER S-303</td>
			<td style="width:81px;">AS ONE</td>
			<td style="width:136px;">1-1133-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">NanoDrop Lite</td>
			<td style="width:81px;">Thermo Scientific</td>
			<td style="width:136px;">ND-NDL-US-CAN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:81px;">TAKARA</td>
			<td style="width:136px;">RR037A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">KAPA SYBR Fast qPCR kit</td>
			<td style="width:81px;">Kapa biosystems</td>
			<td style="width:136px;">KK4601</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

우리는 먼저 애기 장대 성숙한 씨앗을 사용하여 PVP의 최적 농도를 조사 하였다. 총 RNA는 0 %, 0.25 %, 0.5 %, 1.0 % 또는 2.0 %의 PVP를 함유하는 세포 용해 완충액을 이용하여 전술 한 프로토콜에 따라 약 1,000 종자로부터 단리 하였다. 오일 층과 시드 파편 (도 1A)를 피하면서 균질화하고, 원심 분리 후 상청을 회수 하였다. <img alt="그림 1" src="/files/ftp_upload/55008/55008fig1.jpg" /> 도 1 : 세포 용해 완충액에서 최적의 폴리 비닐 피 롤리 돈 농도 추정. 총 RNA는 PVP의 상이한 농도를 함유하는 세포 용해 완충액으로 추출 하였다. (A) 균질화 및 원심 분리 후 씨의 사진. (B) 각 샘플에 대한 파장의 흡광도의 그래프를 나타낸다.<html> <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 단리의 RNA의 양과 순도 1.0 %의 PVP 씨앗으로부터 정제 RNA 다량 분리하는 가장 효과적인 농도였다 파장 흡광도의 그래프 (도 1b)로부터 추정 하였다. 개발 씨앗 실체 현미경 (도 2A, 2B)에서 얼음에 미리 냉장 알루미늄 판에 애기 장대의 과일에서 고립되었다. 씨앗은 얼음에 알루미늄 랙 (도 2C, 2D)에 튜브 스테인레스 스틸 봉과 모터 분쇄기와 균질화 하였다. 2.JPG &quot;/&gt; 그림 2 : 분리 및 애기 장대의 종자를 개발 균질화. 개발 씨앗 실체 현미경 (A)에서 차가운 알루미늄 접시에 과일에서 격리됩니다. 심피는 척추 경 (B)로부터 박리하고, 개발 씨앗 수집됩니다. 씨앗을 얼음 (D)에서 냉각 알루미늄 랙 튜브 스테인리스 유봉 (C)를 사용하여 폴리 비닐 피 롤리 돈 (/ V W) 1 %를 함유하는 용해 완충액에서 균질화된다. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 총 RNA는 새롭게 개발 된 방법 또는 통상적 인 방법을 사용하여 (이하 DAF 라 함) 개화 후 4, 8, 12 일째에 종자로부터 단리 하였다. 농도, A260 / A280 비율 및 A260 /종자 (200)로부터 분리 된 RNA의 A230 비율을 표 1에 나타내었다. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 방법 </td><td> DAF </td><td> RNA 농도 </td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td rowspan="3"> 전통적인 </td><td> 4 </td><td> 72.4 ± 7.4 </td><td> 1.90 ± 0.02 </td><td> 2.27 ± 0.06 </td></tr><tr><td> 8 </td><td> 10.8 ± 3.0 </td><td> 1.50 ± 0.08 </td><td> 1.40 ± 0.17 </td></tr><tr><td> (12) </td><td> 4.9 ± 1.7 </td><td> 1.57 ± 0.10 </td><td> 0.76 ± 0.18 </td></tr><tr><td rowspan="3"> 새로운 </td><td> 4 </td><td> 63.7 ± 2.6 </td><td> 2.10 ± 0.08 </td><td> 2.29 ± 0.03 </td></tr><tr><td> 8 </td><td> 46.3 ± 4.9 </td><td> 2.15 ± 0.05 </td><td> 2.23 ± 0.09 </td></tr><tr><td> (12) </td><td> 41.77; 3.1 </td><td> 2.09 ± 0.04 </td><td> 2.17 ± 0.07 </td></tr></tbody></table> 표 1 : 애기 장대 종자에서 분리 된 RNA의 농도, A260 / A280 비율 및 A260 / A230 비율. 총 RNA는 4, 8에서 약 200 씨앗 (190 206 씨앗)에서 분리하고, 12 DAF는 종래의 방법 또는 우리의 새로 개발 된 방법을 사용하여. RNA의 농도, A260 / A280 비율 및 A260 / A230의 비율을 측정 하였다. 값은 3 개의 독립적 인 실험의 평균 ± SD를 나타냅니다. 결과는 우리의 방법은 8, 12 DAF 씨앗에서 고순도 RNA를 분리 할 수있게 보여줍니다. 총 RNA는 cDNA를로 역전사시키고, cDNA의 용액을 100 배 희석 하였다. 복사 WRI1의 수 (주름 1 : AT3G54320), SDP1 (SUGAR DEPENDENT1 : AT5G04040) 7 &lt;/ SUP&gt; 및 EIF3K (진핵 번역 개시 인자 3K : AT4G33250) 내부 제어 8 등을 측정하고, 그 성적의 상대적인 양은 정량적 PCR 분석 실시간 (도 3)에 의해 추정 하였다. <img alt="그림 3" src="/files/ftp_upload/55008/55008fig3.jpg" /> 그림 3 : 개발 씨앗에서 낮은 수준의 성적 증명서의 정량화. 총 RNA는 우리의 방법을 사용하여 4, 8, 12 DAF 씨앗으로부터 추출하고, cDNA의 용액을 제조 하였다. WRI1 (A) 및 SDP1 (B) 전 사체의 상대적인 양을 정량적 실시간 PCR에 의해 측정 하였다. EIF3K는 내부 대조군으로서 사용 하였다. 값은 3 개의 독립적 인 실험의 SD를 의미 나타냅니다. DAF; 꽃 후 일. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig3large.jpg" target="_blank">그는를 클릭 해주세요</a>이 그림의 더 큰 버전을 보려면 다시. WRI1 및 SDP1 사체의 양이 현상 씨앗 상대적이지만, 상이한 발달 단계에서 종자 간의 발현의 변화를 검출 할 수 있었다. 우리의 방법이 다른 종자에 이용 될 수 있는지 확인하려면 총 RNA는 브라 시카 napus와 글리신 최대의 성숙한 종자에서 분리 하였다. RNA의 농도, A260 / A280 비율 및 A260 / A230 비는 표 2에 나타내었다. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 방법 </td><td> 식물 </td><td> RNA 농도 </td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td> 전통적인 </td><td> B. napus와 </td><td> 7.7 ± 0.8 </td><td> 1.5 ± 0.12 </td><td> 0.75 ± 0.11 </td></tr><tr><td></td><td> G. 최대 </td><td&gt; 9.7 ± 2.3 </td><td> 1.61 ± 0.06 </td><td> 0.69 ± 0.09 </td></tr><tr><td> 새로운 </td><td> B. napus와 </td><td> 143.2 ± 19.0 </td><td> 2.17 ± 0.03 </td><td> 2.23 ± 0.13 </td></tr><tr><td></td><td> G. 최대 </td><td> 152.3 ± 4.4 </td><td> 2.17 ± 0.02 </td><td> 2.30 ± 0.02 </td></tr></tbody></table> 표 2 : RNA의 농도, A260 / A280 비율 및 A260 / A230 비율은 브라 시카 napus와 글리신 최대의 성숙한 씨앗에서입니다. 총 RNA는 브라 시카 napus와 종래의 방법 또는 우리의 새로 개발 된 방법을 사용하여 글리신 최대 종자의 약 100 mg을 분리 하였다. 씨앗은 이전에 분쇄와 액체 질소에 냉각 박격포와 유 봉 대략 균질화 하였다. RNA의 농도 A260 / A280 비 및 A260 / A230의 비율을 측정 하였다. 값은 3 개의 독립적 인 실험의 평균 ± SD를 나타냅니다.

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An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

양 사체 (Transcriptome) 분석에있는 사용을위한 씨앗에서 매우 정제 된 RNA의 분리를위한 효율적인 방법

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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Nanyang Technological University

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JoVE Journal

Biochemistry

9.6K 조회수.  National Institute for Basic Biology. We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

비디오: An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

작은 RNA 정량 비는 코딩을위한 고밀도 지방 단백질의 분리

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

연구

생화학

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

레이저 이외에 Microdissection RNA-서열에 대한 실험 디자인 : 옥수수 잎 개발의 분석 교훈

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

양적 질량 분광법 분석을위한 지질 물방울 절연

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

단백질 및 모 세관 전자 초점 방법으로 그들의 Isoforms의 매우 민감하고 정량적 검출

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

식물에 철 분 함량을 측정 하는 색도계 방법

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

번역 개시 포유류 mRNAs에 복잡 한 대형의 Toeprinting 분석

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 세포에서 Mitoribosome의 신속한 격리

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

생체 내피 세포에서 RNA 격리를 위한 리보솜 친화성 정제(TRAP) 번역

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

유전자 발현 프로파일링을 위한 수정된 방법으로 시리얼 씨앗에서 고품질 전사 데이터 획득

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.