Hi all,
We aim to compare differential mRNA expression between 2 different cell types. We sorted 2 populations of cells by FACS, extract RNA by column based methods, and SMARTSEQ v2 for pre-amplificaiton of cDNA.
Between those cell types, A has very low cell number (150-500 cells). In contrast, B has higher cell number (50,000 cells) but its RNA amount is less. So, I only do RNA quantification for B, because RNA amount in A is too low to be detected by Ribogreen.
For Pre amplification of cDNA, I have some questions:
(1) If the starting RNA amount used for SMARTSeq V2 is different between samples, will it affect the RNA Seq results, if we aim to compare the differential mRNA expression between those samples?
(2) For the pre-amplification of cDNA, will the number of PCR cycles for A and B need to be the same, if I would like to compare the differential mRNA expression between those cell types? If sample A need 20 cycles to get sufficient cDNA, while sample B only need 15 cycles, what should I do? Thank you
We aim to compare differential mRNA expression between 2 different cell types. We sorted 2 populations of cells by FACS, extract RNA by column based methods, and SMARTSEQ v2 for pre-amplificaiton of cDNA.
Between those cell types, A has very low cell number (150-500 cells). In contrast, B has higher cell number (50,000 cells) but its RNA amount is less. So, I only do RNA quantification for B, because RNA amount in A is too low to be detected by Ribogreen.
For Pre amplification of cDNA, I have some questions:
(1) If the starting RNA amount used for SMARTSeq V2 is different between samples, will it affect the RNA Seq results, if we aim to compare the differential mRNA expression between those samples?
(2) For the pre-amplification of cDNA, will the number of PCR cycles for A and B need to be the same, if I would like to compare the differential mRNA expression between those cell types? If sample A need 20 cycles to get sufficient cDNA, while sample B only need 15 cycles, what should I do? Thank you