Guidelines for qPCR

RNA extraction:
1. While extracting RNA be careful on the use of Trizol. Trizol inhibits microRNA (Trizol skews microRNA results). There are two kits which are trizol free – Zymoresearch RNA extraction kit and Qiagen miRNA isolation kit.
2. To avoid frothing of tissue, while homogenizing, I would recommend using “Reagent DX” from “Qiagen.”
3. If the kit contains in-column DNase treatment, then you should use it – saves time.
4. RNA elution could be done in either DEPC water or TE buffer and store at -80°C. If possible, RNA should be aliquot and stored so as to avoid repeated freeze-thaw.
5. RNA should be measured (if thawed again) for determining if there is any degradation. Before measuring RNA, give it a brief spin and pipette it up and down at least thrice so that the exact measurement of RNA can be determined.

cDNA synthesis:
1. cDNA should be made immediately or within a day of RNA extraction so as to avoid freeze-thaw of RNA.
2. After reverse transcription of RNA, dilute and aliquot the cDNA.
3. Store all the aliquots at -20°C and take out one at a time. You can store one aliquot at 4°C for a maximum of 1-2 months.

qPCR:
You have to be extremely careful while preparing the plate and the qPCR machine.
1. Wipe off the bench and all the pipettes that are going to be used for qPCR first with 100% ethanol and then RNAse Zap. You can purchase RNAse ZAP from Sigma-Aldrich, which is cheaper (link below) compared to life technologies.
2. First, make the master mix by adding the qPCR master mix, the primer pair, and water. After, you are done with the primer pair place it back in the fridge or keep it far away.
3. Now take out the qPCR plate from its package and pipette the master mix to the qPCR plate.
4. Take the cDNA out of the fridge and add it to the qPCR plate and immediately cover the plate with optical seal cover.
5. Give the qPCR plate a brief spin down (high speed for 2 min).
6. Place the qPCR plate in the machine and run the program.

Links to products:
I am in no way endorsing the products, be careful with your samples, and I am not responsible for the loss of your valuable samples. I would also recommend the use of DEPC water. These products work best in our research

DEPC water: https://www.carlroth.com/en/en/Chemicals/A-Z-Chemicals/W/Water/Water/p/000000010000562d00020023_en
Reagent DX: https://www.qiagen.com/de/shop/lab-basics/buffers-and-reagents/reagent-dx#orderinginformation
Zymo Research RNA extraction kit: http://www.zymoresearch.de/rna/rna-isolation/routine-rna-isolation/quick-rna-miniprep
cDNA synthesis kit: https://www.thermofisher.com/order/catalog/product/K1612?ICID=search-product
qPCR master mix: https://www.thermofisher.com/order/catalog/product/4385610?ICID=search-product
RNAse Zap: http://www.sigmaaldrich.com/catalog/product/sigma/r2020?lang=de&region=DE

 

 

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