Hello dear Karthikeyan,

First of all,  I would reisolate my sample, because, RNA is very unstable and it will be degraded in any purification method. And you might face some other problems in dowststream applications due to degradation. However, if you have no chance to reisolate, then I advice to use ethanol or isopropanol to precipitate your RNA. If you want to use isopropanol, I advice to wash with ethanol right after. And dissolve your RNA pellet with 25-50 ul of RNAse free dH2O.

Hope it was helpful,

Regards

UA

Hello dear Karthikeyan,

First of all,  I would reisolate my sample, because, RNA is very unstable and it will be degraded in any purification method. And you might face some other problems in dowststream applications due to degradation. However, if you have no chance to reisolate, then I advice to use ethanol or isopropanol to precipitate your RNA. If you want to use isopropanol, I advice to wash with ethanol right after. And dissolve your RNA pellet with 25-50 ul of RNAse free dH2O.

Hope it was helpful,

Regards

UA

Dear Karth

A prominent peak at 230nM and a 260/230 ratio < 1.0 is caused either by phenol/Chloroform/Trizol in the case of manual extraction of RNA or Guanidinium salts in the lysis buffer of column based RNA extraction kits

The object therefore of supplementary purification is to remove the offending contaminant i.e. Phenol/chloroform/Trizol and/or GITC which can inhibit downstream RNA amplification and/or reverse transcription

By removing such contaminants the aforementioned peak is general eradicated; the 260/230 ratio is rendered > 1.0 and in my experience a sample refractory to processing becomes amenable to RT/RNA amplification and successful downstream applications, e.g. qPCR and array work

Generally speaking there are two types of re purification, both of which have worked successfully in my hands:

1. Alcohol/salt purifications like butanol + 3M Sodium acetate or ethanol + 3M sodium salt + 70% wash. Both methods work well and in particular having precipitated your RNA washing with 70% ethanol, preferably 2 x and preferably using room temp 70% ethanol is necessary form removing GITC salt in column based extraction methods; In the case of phenol/chloroform/Trizol extraction re precipitation itself should eliminate the organic solvent although Trizol actually contains GITC as well as phenol/ chloroform so the 2 x room temp 70% wash is still necessary. I would also say that for small amounts of re precipitated RNA adding 1ul of molecular grade glycogen @ 1mg/ml to 20mg/ml to the ethanol or butanol plus sodium acetate mix renders the eventual precipitated RNA pellet more visible, allowing vortexing with 70% ethanol, thereby increasing the washing efficacy and also increases total RNA recovery (an issue if just a few ug of RNA comprise the total sample)

2. In the case of column based re purification methods to remove contaminants causing the 230 peak and rendering the 260/230 ratio < 1.0 if you simply mix your sample to be re purified with an equal vol of 70% ethanol; apply to the column and then follow the standard protocol, in my experience this also works well  

Dear Stuart and Ulvi 

Thanks for the reply. 

I have used manual trireagent isolation method to process my samples. 

Will try with butanol+ sodium acetate+ ethanol wash method and will post results. 

Thanks again

Dear Karth

tri reagent is trizol; that is phenol chloroforn plus guanidinium salts. Therefore you contaminating peak at 230nm are indeed due to one or more of these components bleeding through to the final RNA

Either precipitate with butanol plus sodium acetate OR ethanol plus sodium acetate NOT both. I would personally use 3 volumes of ethanol and 1/10 volume 3M sodium acetate pH 4-5.  For RNA precipitate for a minimum of 1 hour to overnight at -80c; spin for 20 min at 13 000rom at 4C; then wash with 70% ethanol. I can provide more details of the wash if you wish

Dear Stuart 

Thanks for valuable suggestion.

I have 10ul of RNAs, stored in -80 C. So it means add, 30 ul of 100% ethanol and 1ul of 3M sodium acetate and 10ug- 20ug glycogen, After incubation in -80 C for overnight. Spin and wash with 70% ethanol.  (please correct me, if there s any mistake).

Dr.Stuart , indeed adding any more details will improve the process.,

And i read some of your comments (reg RTPCR primer optimization) and seen you have added PDF link, which is so good to have for any researchers.  Same way, if you have any detailed link reg RNA isolation and trouble shooting,It will be great to have.

Thanks 

II prefer a commercial isolation kit instead of TRIZOL+precipitation or other alternatives. I have never tried magnetic beads, which seem to produce really nice results. I usually use a column based method, such as RNeasy.

In my experience, TRIZOL-like methods tend to isolate more RNA, but the resulting purity is usually far from good. Moreover, using several reagents that are not included in a RNAse free kit, significantly increases the risk of RNA degradation, which is the paramount problem when working with RNA.

On the other hand, column based methods isolate less RNA, but purity is usually the best possible.

If you are worried about low RNA concentration when using column based methods, I would recommend a micro kit (eg: RNeasy plus micro), which allows ellution in as little as 14 ul of NAse free water. Plus, I recommend adding the elution buffer (or H2O) and let the colunn equilibrate for at least 5 minutes, then elute, pick the eluate and put it back in the column for another 5 minutes, then elute. This extra steps and incubations significantly increase RNA concentration.

For maximum RNA recovery with column based methods, perform the same steps as above, but after the first elution, put a new elution buffer in the column, leave 5 mins and elute. This steps significantly increase RNA yield, although concentration will be lower than in the method described in the previous paragraph.

Hope it helps.

Dear Karth

I have provided a link to a previous question describing in detail what I you need to do. Take a look at the whole thread, but in particular pay attention to my first long and detailed answer which in effect is a short hand SOP for how you precipitate with ethanol, sodium acetate and (preferably) sodium acetate

In terms of your actual sample volumes:

1. Yes, with 10ul of RNA I would add 90ul of water and then 3 volumes ethanol for the resulting diluted sample, i.e. 300ul of ethanol; To that add 10ul of 3M sodium acetate pH 4-5. With just ethanol and salt incubate over night @ -80C; 1 hour @ -20C the next day (to reduce viscosity) and then spin as described on my detailed former answer

2. For a larger more opaque pellet and better over all recovery, to the above you could add 1ul of 1mg/ml to 20mg/ml molecular grade glycogen. In this case you only need to incubate for 1 hour @ -80C and then spin directly. However, if more convenient, i.e. if you perform at the end of the day, you can incubate over night (it wont do any harm)

For Future optimal trizol (Tri Gene) extraction I have SOP but not to hand; I can try and dig out for you if you wish. In the mean time, consider these salient points:

A. Add an equal volume of Trizol to your sample and VORTEX for 1 minute. Make sure you sample is at least 100ul (100ul to 500ul ideal) and therefore add 100-500ul of trizol

B. Now spin for 5 min @ 13K (preferably) @ 4C (if using 1.5ml eppendorf or 2ml screw cap tube; the latter is preferable) to ensure good phase separation

C.  Remove sample from the top of the upper aqueous phase GENTLY with a p100 tip

D. DO NOT go all the way down to the interphase as this is where much of your denatured protein emulsified lipids and genomic DNA resides

E. If your bottom trizol layer is translucent, add the removed aqueous layer to an equal volume of 24 parts molecular grade chloroform: 1 part iso amy alcohol (IAA). This mixture (as opposed to 100% chloroform) assists with selective removal of genomic DNA from the final purified total RNA prep. For TC cells or  a small (ug) mass of tissue 1 x trizol (trigene) extraction is probably sufficient

F. For larger tissue masses and/or where following your spin @ 13K for 5 minutes results in an opaque lower layer repeat the Tri gene extraction in order to remove more lipid and protein   

G. Also keep in mind that the lower organic phase during initial extraction will remove 'volume' from the upper decanted aqueous layer: Thus, I will always make sure my aqueous phase after extraction has a volume of 100ul, if necessary by supplementing with water by chloroform: IAA extraction

H. Vortex again for 1 minute at 13K

I. Now spin for 5 minutes as before

J. Now remove the upper aqueous phase carefully as before, decant to a new tube, add an equal volume of isopropanol and spin for 20 minutes @ 13K @ 4C; Residual salt, especially with isopropanol precipitation, is sufficient to precipitate your RNA. Also, DO NOT chill incubate with isopropanol as this will encourage precipitation of salt into the final RNA pellet. Preferably add 1ul of 1mg/ml to 20mg/ml glycogen to this mix before spinning 

K. Remove supernatant and vortex with 1ml of room temp 70% ethanol 

L. Spin @ 13K for 5 min; Remove 70% ethanol; spin again for 1 min; remove residual ethanol; dry pellet initially on ice for 15 minutes and complete @ RT for ~ 10 minutes 

Resuspend in water or TE on ice for ~ 1 hour intermittently pipetting up and down 

https://www.researchgate.net/post/Why_am_I_getting_aqueous_phase_contamination_with_Trizol

https://www.researchgate.net/post/how_to_make_concentration_high_of_DNA_extracting_from_PCR_Product#view=578c9799ed99e132b905cde3

Dear Stuart and Iker

Thank you for the reply.

Iker, we have tried qiagen kit previously, but the yield and as well as purity (esp 260/230) was less than the manual tri reagent method.

Stuart. I m grateful to you for your valuable information. Will get back to u with the outcome.

Dear Karth

just out of curiosity what is your source material 

I am having problems adding further comments to your point so will try again tomorrow 

Dear Stuart

Source- Cells isolated from muscles and grown up for three weeks (in  specific till Passage 2).

I have used approx 5 x 105 cells for RNA isolation, started with 0.5 ml of trireagent

Dear Karth

generally speaking tri reagent or tri gene deal better with RNA purified from mammalian tissue as compared with standard Qiagen RNEasy columns; There are more specialists columns you can buy that deal with connective tissue and issues with lipids in particular that are a characteristic of nascent tissue. What is more standard Qiagen RNEasy miniprep columns have a saturation capacity of 100ug RNA/DNA so that a T-75 flask containing 10(^7) cells can saturate such columns culminating in no elution of RNA; I have had this happen to me but in your case such low cell numbers shouldn't result in that particular problem. Nevertheless, Trizol does tend to process tissue and issues relating to complex carbohydrates and lipids characteristic of mammalianl tissue/cells more effectively that standard silica based columns (whether Qiagen or a technical clone by another manufacturer

That said there are some advantages to columns particularly in terms of eradicating gDNA and thus the best RNA is often obtained by intial extraction with Trizol/trigene and chloroform followed by supplementary clean up with a column. This invariably has given me the best quality (260/230 > 1.0-1.5 and 260/280 ratio > 1.8) and quantity of RNA. Specifically:

1. Perform Extraction with Trizol/Trigene as already described, i.e. vortex hard for 1 minute and spin 13K in screw cap tube or 1.5ml eppendorf @ 13K for 5 minutes to procure good phase separation (reducing this time can result in contamination of the aqueous layer containing RNA, some gDNA and salt with the interphase layer)

2. Carefully remove the upper aqueous phase from the top down and deliberately omit the last few ul so as not to disturb the interphase layer (containing some denatured protein - much is in the lower organic phase - ,coalesced lipid, complex carbohydrates and gDNA)

3. Decant this onto Chloroform:iAA (24:1) making sure to supplement your aquoes phase with Milli Q water if volume < 100ul

4. vortex and spin as already described

5. Remove upper aqueous layer and rather than performing isopropanol precipitation @ RT add an equal volume of Room temp 70% ethanol to your extracted material and add to a standard qiagen column

6. Now carry out the standard RNEasy protocol (which is principally designed, in view of prior extraction, to remove salt and gDNA) with the following modifications

A. Perform on column DNASe I digestion: Even by setting the nano drop to RNA this can increase the reading and so ultimately you will actually end up putting less RNA than you think into the next step

B. Having added the 2nd wash buffer plus ethanol to your column wait 1 minute before spinning through. Now repeat this ethanol wash buffer step: The ethanol in this second wash buffer is designed to remove GITC present in the first wash buffer which is designed to complete denaturation of any  on column proteins. This step by more effectively removing GITC will render your 260/230 ratio > 1.0 and result in more 'functional' RNA (GITC denatures protein and can thus inhibit enzymes like reverse transcriptase)   

C. Having spun the column dry following your 2 x ethanol washes, add 30ul of Milli Q (molecular grade) water; wait 5 minutes then spin. Now take the recovered eluate; putback on the column; and a further 20ul of Milli Q water; wait 5 min then spin again: This double elution with incubation increases RNA recovery 

Dear Stuart

Thank you so much for the protocol. It is worked.

I run 15 samples following your protocol. Except one sample all others, both 260/280 and 260/230 values were increased. Most of them shown clear peak at 260 with 260/230 value approx 2 (These samples were less than 1 260/230 previously).

One sample 260/230 value is 1.20 and the peak is between 265 to 270 range. I have tried two times sodium acetate precipitation but could nt help. Will you please comment if you have any suggestion.

Best , Karthikeyan.R

Dear Karth

• glad the protocol helped for most samples
• With single sample the 260/230 ratio > 1.2 although not as good as most approx to 2.0 is still fit for purpose, i.e. > 1 indicates low levels of GITC
• For that single sample where ppt hasn't helped my only remaining suggestion would be to mix with an equal vol of 70% ethanol and then purify with a Qiagen RNEasy column