Which RNA extraction method would you recommend for RNA-Seq of snap-frozen fetal murine tissues? We will be doing Lexogen QuantSeq. Considering Qiagen RNeasy (good, but pretty expensive) or Thermo GeneJet RNA (much cheaper but we have less experience with it). I heard some kits leave so much chemical contamination that library preparation doesn't work well.
I think RNA is first small step and rest hugh and expensive steps are RNA-seq and analysis all depends upon quality of RNA. RNAeasy kit I have used it gives good quality of RNA. . It is worth spending a bit more money for RNA isolation.
Mikael Niku
I have prepared samples with Qiagen RNAeasy Kits, Trizol method, and Thermofisher's Magjet. Every methods has it pros and cons associated with it and depending on your number of samples. If it is a smaller sample size (less than 40) I would honestly do Trizol isolation, it is the slowest but usually yields the best results for quality and concentration. If it is a large amount of samples I would do either Qiagen RNAeasy or MagJet. The main thing with any RNA isolation method is cleanliness and checking every step for contamination of gDNA. So for RNA seq, make sure you have RNAse Away on your pipets and everything that is going to be used to RNA prep. Afterwards, run samples on a Bioanalyzer to check the quality score and to get a concentration (Nanodrop is not recommended for quality assurance with sequencing samples) as well as checking for fragmentation. I would also run my RNA out on a nondenaturing gel before submitting samples to the bioanalyzer (Chips for Bioanalyzer arent cheap). I have attached my protcol for my RNA nondenaturing gel.
Hello! I really like to use TRIzol (or QIAzol) and then the QIAgen RNeasy (mini or micro) kits. I prefer a chemical extraction. There is a protocol in the Appendix of the QIAgen RNeasy manual for this method. I always use the DNase that comes with the RNeasy kit. Be careful with DNase that is in the RNeasy kit, because after hydration it is shock sensitive. I agree that is is very important to be careful about genomic DNA contamination. I bought some long and thin tubes from Thermo, with adaptors, for a refrigerated microcentrifuge so as to avoid the genomic DNA at the interface of the phenol/chloroform extraction when using TRIzol. Lab hygiene and engineering controls are very important for RNA work. In an isolated area that is not used for PCR (!) I use Eppendorf tubes that are "DNA LoBind", aerosol blocking pipet tips, RNAse zap wipes and spray. Its good to keep your RNA equipment separate from your regular lab equipment. The QIAgen kit will have clean tubes, if you don't want the LoBind ones (they are nice though). I wipe down the centrifuge in between separating the phases (TRIzol part) and isolating the RNA (RNeasy part). This method is also flexible, if you isolate really small amounts of total RNA, as you can use glycogen and Lithium Chloride precipitation with TRIzol and the long skinny tubes. We use the full size LoBind tubes after we separate phases. We also have a speed-vac for drying nucleic acids. These are old, but good techniques that don't need kits. Check Molecular Cloning, third edition, Volume 3 Appendix A.12. With LiCl precipitation you should still do the DNAse procedure. I don't run the gels usually, but I think they are a good idea and I may start, but I usually just spec the sample on the Nanodrop. If the A260/A280 ratio is over 2.0 and the trace looks good, then I send it off for the Bioanalyzer. Caution! If you use Glycogen the absorbance at 260 nm will be affected and the A260/A280 ratio is meaningless, just send to the Bioanalyzer. We don't make libraries from RIN less than 7.0 and we prefer 10. Good luck!!
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