Hi all,
I FACS sorted 50000 cells and Isolated RNA from them using RNAeasy plus micro kit. Isolated RNA showed 260/280 ratio of 2.08 but, 260/230 ratio is 0.1. Conc. was 3ng/ul. we dont have bioanalyzer so didn't calculated RIN value. I converted this RNA to cDNA and ran qPCR for 18s with minusRT as control.
I am getting Ct of 20 for 18 s.
My question to you all is whether Ct of 20 for 18s tells anything about quality of RNA?
Whether This RNA can be subjected to RNA seq?
best regards,
Arshed
If you see amplification of 18s starting from 50000 cells then I'd say you're in a good shape with your RNA (and cDNA) anyway; well done. Unless you have to run RT-PCR for very small messengers detection, very low represented messengers, miRNA or smallRNAs, then let's just load the plate and hope for the best. As far as you see specific amplification in the 10 to 30 Ct range, you could always run your RT-PCR products on agarose gel for band purity/size or send it for sequencing if you're not really convinced about your results.
Hi Arshed,
The Ct doesn't really tell you anything about the quality of the RNA. You can make inferences that a low Ct value from a high/medium copy number target means the quality is good while a high Ct value means the RNA is degraded but other factors could be at work. (for example, your poor A260/230 indicates solvent impurities that could interfere with both RT and PCR, so a high CT value may not be indicative of bad RNA). Ct of 20 is probably fine.
With that in mind, since you are planning on running RNAseq, I would definitely recommend trying to clean-up that solvent (probably ethanol or guanidium) prior to spending the money on RNASeq. RNEasy type kits or mag beads are less stress inducing than phenol-chloroform...
--Matt
A260/230 is too low, which means impurity as well as concentration of 3ng/microlit is too low. I would suggest increase the cell number to atleast 4times.
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