I'm dealing for the first time with RNA, with the final aim of performing RNA-seq analysis to estimate the differentially expressed genes of a gram negative bacteria during a certain process.
I'm now trying to estimate the scale of my culture to be able to harvest enough cells to obtain a good RNA sample, of good quality, and using the RiboPure kit (Ambion) for RNA extraction.
After my last trial, all my samples showed a good RNA concentration, 180-620 ng/ul, and good A260/A280 rations, >2.0. Most of the A260/A230 were also good, 1.9-2.2, but some of them showed a lower ratio, of 1.3-1.4.
Do you think RNA-seq could be possible for these samples? Are my preparations of quality enough or should I repeat the RNA preparation for those samples showing a lower A260/230 ratio?
Thank you very much in advance.
Hello, I have no experience in RNA-Seq analysis of bacterial transcriptome (I only study eukaryotic transcriptome), but I guess the 260/280 and 260/230 look good (even 1.3 and 1.4). But these ratios reflect the purity, maybe you have to check integrity of your RNA (using bioanalyser).
Hi
Unfortunately Nanodrop does not say anything about the integrity of your RNA and that is the most important thing one must look out for to do RNAseq libraries. If with bioanalyser, you should get a RIN value of atleast 7. If you do not have access to a bioanalyser, you could run the sample in the gel and see if you get 2 clear rRNA bands without much degradation bands at the bottom.
Hi
You must have to check the RNA quality for any RNA-seq experiment on the gel and bioanalyzer (Agilent RNA 6000 Pico Kit). On bioanalyzer your RIN value should be more that 6.5 or on gel your 23:16s ratio should be 2:1. These are the very essential criteria for any RNA-Seq experiments.
Hi, Cristina,
- the Agilent-provided RIN value will help you in testing the integrity (should be >~7).
RIN is favourable, but if this Bioanalyzer is difficult to find for some reason, I'd like to add is that you can still rely on the RNA integrity test by old-fashioned gel electroporesis, by which degraded RNA can be easily spotted. The key is then to have a good positive control.
- the OD will give you some measure of purity (your values look reasonable)
You mentioned different treatments. I expect that you use biological replicates. The drop in od260/230 may only be something to be worried about when this drop is correlated to treatment.
Hi Cristina,
The RiboPure kit works very well with gram negative bacteria, however as already stated by others, you have to check the RNA quality by Bioanalyser (I get jusually RIN of 9-10) for RNA-seq where the RNA quality is very important.
Working with bacterial RNA don't forget that for RNAseq you have to get rid of rRNA before sequencing and again the test with Bioanalyser is necessary to monitor that picks of rRNA have desapeared.
Hi Christina,
especially when you are dealing with RNA for the first time, you should really check the RNA quality (Gel/Bioanalyzer) and not only purity (Nanodrop/photometer). As you are dealing with cell cultures I would expect a RIN of 9-10 using the bioanalyzer or absolutely no sign of degradation on a gel. A RIN of 7 is acceptable if you are dealing with RNA from tissues (or even a RIN of 6 in problematic cases like pancreatic cancer tissue), but a RIN of 7 with cell culture experiment indicates problems with the RNA isolation (IMHO).
Hi Christina,
I think you need to look at the overall base sequence quality, GC content and sequence duplication[over represented sequences]. You can use NGStoolkit and FastQC for this purpose.
Thank you very much for all your answers. They will help me a lot.
Hi Cristina
I fully agree with other colleagues: it is necessary to check all yours samples on a bioanalyser. If you don't have such an analyzer in your lab, check in the labs around, more and more lab have such facilities nowadays. They may of course ask you to pay for the chips (12 samples a chip) you'll be using.
Otherwise, if you plan to send directly your RNAs to a sequencing company (i.e. you do not prepare your cDNAs yourself), the RNA quality of your samples will most probably be checked before the synthesis of cDNA banks.
Agreed with the above - you can use the Nanodrop to determine your concentration (which you need to know), but a bioanalyzer (Agilent Pico 5000 card works great) is necessary to determine your RIN. All that said, with a fresh RNA prep using an Ambion kit, your RIN is going to be 9+, more than likely. It's very probably not even a concern. Now, if you're pulling these from long-term storage, I can see that you may be worried. On a fresh prep in a nice RNase free environment, your concerns should be minimal. These samples will be perfectly suited for RNA-Seq.
Hi guys,
Do RIN value >6.5 implies that the RNA is free of gDNA contamination? Can the Bioanalyzer detect gDNA?
Thanks
Generally not - it implies more that your RNA quality is sufficient for sequencing library construction. A BA is able to detect gDNA, but you have to use the DNA chips for it. DNA 12000 is a good one for this.
- Badges
- Science method