The overall goal of this procedure is to allow for rapid and reliable automated setup of QPCR based arrays. This is accomplished by first preparing the primer sets for the array and setting up the sample master mixes to be screened. Next either an automated robotic PCR setup or a matrix electronic pipette system is used to accurately Eloqua sample master mix and primer sets into each well of a 384 well plate.
Once prepared, the 384 well PCR reactions are run with the light cycler four 80 cyber based PCR program. The final step of the procedure is to analyze the QPCR data and determine relative expression levels for each primer pair tested, each of which targets a specific mRNA. Ultimately, results can be obtained that show changes in the expression of large sets of mRNAs.
Specifically developed arrays can therefore target whole pre micro RNA profiles, specific pathways or molecules key to viral infection through QPCR based profiling. Hello, I'm Dirk DMA from the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. Today we will show you how to set up real-time QQPC rra and we will show you two methods.
One, using irregular pipette and one using a robot. We use this technology to measure pre microRNAs and mature microRNAs in human tumors. The demonstration today will be done by Pauline Chu and Kristen bu.
So let's get started. To begin this procedure, retrieve primer plates containing 186 primer pairs in a 96. Well format at 0.5 picaMoles stored at minus 20 degrees Celsius.
After thawing the plates out at room temperature, vortex and centrifuge them briefly. Also thaw cyber green two times PCR mix at room temperature for setup of each 96 well primer plates four master mix tubes will be needed. Prepare the master mix by combining four microliters of cyber green mix three microliters of PCR grade water and 10 to 20 nanograms of sample DNA or CDNA per reaction in each of four two milliliter einor tubes.
Each tube should contain enough master mix for approximately 100 reactions, allowing excess for pipetting waste. Vortex the einor tubes to mix. Begin setup of the precursor micro RNA assay using the freedom Tecan EVO robot with initialization of the robot and loading of the EVO wear program.
Then flush the robot three times with 30 milliliters of system liquid to clear the system of air bubbles that will interfere with pipetting accuracy. Next, set up the robot platform to include a 384 well plate and 96 well primer plate. The master mixes a trough containing 2%bleach, a filled system, fluid container, and an empty waste container.
Begin the automated robot run by selecting run twice at the end of the program. Remove the 384 well plate and seal with light cycler four 80 ceiling foil. Then centrifuge the plate.
Briefly place the sealed 384 well plate in position one of the hotel. Then repeat this process for primer plate two with new master mixes and a new 384 well plate. Place the resulting sealed 384 world plate in position two of the hotel.
Open a new EVO wear program for loading the light cycler from the hotel and set the cycling parameters. Then select run twice. Freedom Evo will automatically load each plate into the light cycler and run the cyber green one HRM cycling program.
Once complete. Review and analyze the results as described in the written protocol as an alternative to using the robot. A matrix electronic multichannel pipette can be used to begin place the contents of the master mix tube one into a reservoir.
Set the electronic pipette to aspirate 16 microliters of master mix with two eight microliter dispense steps followed by a purge step. After setting the pipette aspirate 16 microliters of master mix and begin to dispense into a 384 wheel plate. Dispense the first eight microliters into every other row in column one using rows A to O, leaving the next column empty.
Dispense the second eight microliters into the same rows of column three. Then purge over a waste container and dispose tips following this pattern of leaving alternate rows and alternate columns. Dispense across the plate for five more cycles to pipette master mix two.
Utilize the same rows as master mix one, but begin with column two. Repeat this procedure for master mixes three and four, beginning with columns one and two respectively, but this time using rose B to P.Her quoting of the primer plate. Set an electronic pipette to aspirate two microliters and dispense two microliters with a purge step to follow.
Transfer two microliters of primers from column one row A to H of a 96 well primer plate to every other rowing column one of the 384 well plate purge over a waste container and dispose tips. Repeat this pattern for quatt primer for the three remaining master mixes of columns one and two. Then continue this process for columns two to 12 of the 96 well primer plate moving over every two columns in the 384 well plate for each primer column.
Next, seal the plates with a light cycler ceiling, foil and centrifuge. Briefly following centrifugation. Place the plates into a light cycler four 80 and cycle the plates using the cyber green one HRM detection format.
Using the same cycling conditions as before, repeat this procedure using primer plate two Eloqua into a fresh 384 well plate using freshly prepared master mixes. Precursor micro RNA signatures emerge through profiling using a novel QPCR based array. The average delta CT as normalized to U six was calculated and standardized values were loaded into array.
Minor software yielding three distinct clusters displayed as heat maps. The relative expression of each precursor micro RNA is shown red and blue represent an increase and decrease in relative expression respectively. The color intensity indicates the degree of expression change.
The majority of precursor microRNAs underwent small insignificant changes as expected. However, a small portion of precursor microRNAs significantly increased throughout the time course experiment. Additionally, the relative level of some precursor microRNAs decreased dramatically.
In some instances. They also cluster together in the heat map analysis. Depending on the experiment clusters may emerge that are dependent on viral infection, cell type specific expression of precursor microRNAs or precursor microRNAs that are regulated by a common molecule or signaling pathway.
The average cycle thresholds or cts from a Posey sarcoma associated herpes virus negative sample running quad duplicate are shown. Importantly, Kaposi's sarcoma associated herpes virus latency associated nuclear antigen or KSHV Lana was not detected in this sample by the highly sensitive QPCR array. However, both the internal control U six and let seven a a highly expressed human precursor micro RNA, were expressed at detectable levels.
Finally, as expected, the negative control of PCR grade water did not yield A-Q-P-C-R product. A melting curve analysis for two different primers. Using the same sampling duplicate is shown.
It is clear that the melting temperature is different for the two primer pairs, but the sample is melting at the exact same temperature for each replicate. Signs of a bad or potentially contaminated sample may include several melting peaks suggesting the presence of two different sources of input. We have just shown you how to set up a real-time QPCR experiments using a robot.
It's important that you include all the controls and as many housekeeping genes as possible. So thank you for watching and good luck with your experiments.
