Sample preparation for sequencing
Hello all, The subject of my thesis is the evaluation of gene expression differences between two different species of mammals in a type of cancer. And six samples in each group of case and control are collected separately. At first I have two questions. What is the best protocol for extraction of RNA from PBMCs samples that are collected and stored at -70 for a while? To generate data, and studying the expression level, if it is better to sequence sample by individuals or by pooled samples? Please guide me with your experiences and suggestions
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fereshteh •
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If the cells have been properly kept at -70/-80°C you should be able to extract DNA/RNA without any trouble decades after freezing. Classical column based kits (such as Qiagen and others) are often the easiest for beginners but you could also go for Trizol (RNA) and phenol-chloroform extraction (DNA). Forums such as researchGate are probably more appropriate for this kind of question. Try to have enough biological replicates per condition for your experiment so sequencing samples by individuals will probably be the best.
Dear Fereshteh, Hi
you can use Trizol (Invitrogen, USA) for your RNA extraction.
I think it is better to not pool your samples. Also please have a look at " to pool or not to pool".
~ Best
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