Hi all,
I have done some RNA-seq on relatively large samples (cell count wise) before, so I never really had to run bioA on unpurified samples before.
Now, however, I'm starting to sort low number of cells (100-1000) into lysis buffer (5uL), and I would like to quantify the RNA and/or the cDNA I get from a TSO RT (like SMART-seq2 or SMARTer seq). Or, I would at least like to get some idea of RNA integrity and cDNA sizes
I tried to run the whole lysate on a RNA pico chip (so the RNA is in lysis buffer), and the 28/18S peaks came out weird (attached). The peaks seem to come out at much larger sizes, and not exactly consistent from sample to sample either. Is that due to the salt in the solution? or is that just mechanical error?
Also, how should I run cDNA on the bioA? Some people tell me to run it on an RNA chip, since cDNA (after 1st strand synthesis) should be single stranded. Do I also need to treat the sample with RNAse H? and how about the carry over RNA from the 1st strand reaction? (assuming if I don't want to purify my cDNA out of the 1st strand buffer, since I am assuming the concentration is low)
Any help would be greatly appreciated!
I have done some RNA-seq on relatively large samples (cell count wise) before, so I never really had to run bioA on unpurified samples before.
Now, however, I'm starting to sort low number of cells (100-1000) into lysis buffer (5uL), and I would like to quantify the RNA and/or the cDNA I get from a TSO RT (like SMART-seq2 or SMARTer seq). Or, I would at least like to get some idea of RNA integrity and cDNA sizes
I tried to run the whole lysate on a RNA pico chip (so the RNA is in lysis buffer), and the 28/18S peaks came out weird (attached). The peaks seem to come out at much larger sizes, and not exactly consistent from sample to sample either. Is that due to the salt in the solution? or is that just mechanical error?
Also, how should I run cDNA on the bioA? Some people tell me to run it on an RNA chip, since cDNA (after 1st strand synthesis) should be single stranded. Do I also need to treat the sample with RNAse H? and how about the carry over RNA from the 1st strand reaction? (assuming if I don't want to purify my cDNA out of the 1st strand buffer, since I am assuming the concentration is low)
Any help would be greatly appreciated!