Hello! I have read in an article that RNAlater can be used for RNA storage once it has been extracted. BUT they don't mention how to use it!
...If RNA has to be dilute in it so, when c-dna synthesis is required, the re-precipitation must be made or can be use directly in transcriptase reverse reaction? thinking in inhibition of transcriptase, I consider it would be difficult.... maybe someone who has experience in it can answer me!
Thanks!
My lab is using RNAlater as well, however, from the handbook of RNAlater, it mentioned that it is used to stabilize RNA from sample for storage purpose. What my lab practice is, while going for field sampling, samples were placed in RNAlater, once the samples back to the lab, we will use commercial RNA kit to extract and purify RNA, the purified RNA will be stored in buffer solution (come with extraction kit) and store at -80c. The RNA can be use directly for cDNA synthesis, but avoid too much freeze and thaw cycle of RNA, you may divide samples into small volume aliquots and freeze. Hope this information may help.
Hello, we are doing the same in my lab, I store bacterial cells in RNAlater before extraction, and total RNA extracted on buffer solution that comes with the extraction kit, then -80°C.
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Hi Paul, are you performing qPCR from your samples, or RNA-Seq?
How long do you normally store RNA extracted at -80°C before analysis?
Thank you for your answer
@ Cristina Andrés Barrao, my lab perform 2-step PCR and qPCR from stored RNA after days to months.
In my previous lab, stored RNA were used in irregular time, from weeks to years (tried 5 years), depends on the project. That's why RNA is aliquot in few tubes. The performance is still well.
Your purified RNA could be stored in RNALater, but you will not be able to use it directly in an enzymatic reaction. It contains about 70% ammonium sulfate (along with 18 mM sodium citrate and 14 mM EDTA) at pH 5.2. The very high ammonium sulfate concentration would inhibit enzymes if carried over into a reaction, so you would need to buffer exchange by some method (centrifugal ultrafiltration or re-purification) which would be inconvenient if you have other means to store the RNA (in a simple 1mM sodium citrate buffer, pH 6.4 frozen at -80°C, for example).
RNA later contains high concentration of sodium citrate, EDTA and ammonium sulfate which if you want to use it directly in any PCR reaction, you would fail. However, A column exchange chromatography to get rid if these components is necessary before any RT-PCR.
Regards,
GCK
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