I am extracting RNA from fat bodies tissues using the Trizol protocol. However, the starting material is of low amounts (10µl volume of extracted fat bodies of a small insect). I would like to use the extracted RNA for RNAseq, but it seems that it won`t pass the quality check for BGI. What can I do to increase the concentrations and the quality? I have attached a report from BGI for your reference.
Hi Louis for RNA seq it is very crucial that cDNA you transcribe from your RNA samples should be of higher quality, then even your low yield and low quality of RNA will show promising results when you will prepare the cDNA libraries from such samples. If you are performing mRNA seq, not the whole transcriptome RNA seq then RIN values and 28s:18s ratios won't matter much because it is merely an assumption that you have good RNA, but you cannot figure in fact the quality assessment of your potential mRNA content within your samples out of the total RNA present there, and if you are also not performing any poly A enrichment then your chances are still quite low to capture a reasonable amount of mRNA. A very good RNA quality and quantity may generate poor cDNA depending on the mRNA content and its integrity in the samples, and I have personally observed this numerous times. So in my experience, if you use the following cDNA kit, it will solve all of your problems in terms of having low or bad quality libraries and your RNA seq would run fine. But, along with this, I would highly prefer to focus well and optimize also your library preparation protocol, as more often the actual problem lies in this phase. You need to optimize your protocol as per your sample nature and the type of reverse transcription protocol you are using on your RNA templates.
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Regards....
Jawwad Ahmad Thanks for your reply. The samples are for RNAseq to analyze gene expression profiles. Unfortunately, I am not doing the sequencing in-house but rather outsourcing it to BGI, China. So I have to ensure I obtain RNA of good concentration and quality.
Hi Louis,
If you are looking for the quality you should test the purity first by using the nanodrop and it makes sense you can't send the RNA for RNA sequencing before testing the purity and also check the quantity of RNA.
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