Hello,
For the project I am currently involved in, I am in need for a cheap, non-toxic, pH 7+, non-disruptive buffer for RNA stabilization of bacterial cells (applied to a large environmental sample for subsequent manual cell collection).
RNAlater stabilization reagent is too saline and my cells start floating, so I cannot collect them anymore (the procedure is quite specific).
So I was wondering if anyone could tell me how to prepare RNAprotect from Qiagen. Unfortunately I cannot afford to buy it.
Thanks!
Artur
RNAprotect comes in two different versions: one for eukaryotic cells (RNAprotect Cell reagent), and one for prokaryotic cells (RNAprotect Bacterial reagent).
According to safety data sheet and other sources by Qiagen, it seems that the compositions of the products are the following:
RNAprotect Bacteria Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium bromide (TTAB)
- 200 mM tartaric acid
- NaOH to pH 4
RNAprotect Cell Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium oxalate (TTAO)
- pH 6-7
Dear Artur,
You can use the RNA Later. Good Luck.
http://sfg.stanford.edu/RNAbuffer.pdf
Or just store the bacteria in glycerol (-80*C) stock and extract RNA later.
https://www.addgene.org/protocols/create-glycerol-stock/
Preparation of RNAlater:
- First, one should prepare or obtain the following stock solutions and reagents: 0.5 M EDTA disodium, dihydrate( 18.61 g/100 ml, pH to 8.0 with NaOH while stirring); 1M Sodium Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to dissolve); Ammonium Sulfate, powdered; Sterile water. In a beaker, combine 40 ml 0.5 M EDTA, 25 ml 1M Sodium Citrate, 700 gm Ammonium Sulfate and 935 ml of sterile distilled water, stir on a hot plate stirrer on low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH5.2 using 1M H2SO4. Transfer to a screw top bottle and store either at room temperature or refrigerated.
- https://patents.google.com/patent/EP1657313A2/en
Absolutely do not use RNA Later. DEPC will not help, glycerol can be a growth media for some unfixed bugs. Ethanol, DTT, DMSO have been used by others on the past.
RNAprotect comes in two different versions: one for eukaryotic cells (RNAprotect Cell reagent), and one for prokaryotic cells (RNAprotect Bacterial reagent).
According to safety data sheet and other sources by Qiagen, it seems that the compositions of the products are the following:
RNAprotect Bacteria Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium bromide (TTAB)
- 200 mM tartaric acid
- NaOH to pH 4
RNAprotect Cell Reagent:
- 4% (w/v) Tetradecyl-trimethyl-ammonium oxalate (TTAO)
- pH 6-7
One added comment... RNA later is not, and I repeat, NOT for cellular preps. It is strictly for solid tissues and acts as a traditional salting out reaction. RNA later can not be used in cellular preps because it has a specific gravity of 1.2 and will cause cells to float during centrifugation. So you'll never get your target cells... Or at least the live osmotically healthy cells.
I have a follow-up question to that:
For a dual RNAseq in which RNA of infected cells and bacteria are collected together, which reagent should I use?
I guess RNAprotect Bacteria Reagent would be able to release and stabilize both eukaryotic and prokarytoic RNA?
Thanks
Sven
Never use RNALater. The SG is 1.22. All live microbes will float, all dead will sink. The RNAprotect is TTAB. It is not a "buffer" as you require. Are you sure you need a "buffer" or are you looking for a preservative. Two very different things.
Scott Tighe
Group Leader
Extreme Microbiome Project
Dear Sven. I have addressed Qiagen with the very same question. They replied that RNAprotect Cell Reagent is recommended for "complex" samples, while the Bacteria Reagent is recommended for bacteria only.
Hy everybody,
I am looking for a reagent that would stop gene expression during harvesting of microalgae. Did anybody tried home made "RNAprotect" on eukaryotic microorganisms ? Any clues about the impact of harvesting at 4°C on cold gene expression?
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