Tzu, I wish to verify the information you provided. Firstly, you have provided a photo of your TAE gel displaying your resolved RNA samples. Secondly you have stated your A260/A280 ratio as 1.9 -2.1 and thirdly that you extracted your RNA using the phenol/ChCl3 method. Your question is about the material in the wells and the integrity of your RNA.

A few additional bits of information are required. 1. which of the phenol/chloroform methods did you use? Did you use the acidic phenol/CHCl3 method? (this is relevant to whether the high molecular weight in your lanes are genomic DNA or aggregated RNA.

This brings me to the 2nd information I require. Did you denature the RNA sample prior to running on the TAE gel.

I have resolved RNA on agarose gels with different buffer systems including TAE. You do not have to resolve your samples on formaldehyde or other denaturation gels for good resolution. I have obtained resolution on nondenaturation gels equivalent to that obtained with denaturation formaldehyde gels as long as I prepare the RNA samples in denaturation conditions.

(final concentrations: 50% formamide, 3.7 % formaldehyde, 1X (running buffer ,in your case 1X TAE) 1X dye solution, I also include EtBr to a final concentration of 10ug/ml, therefore I do not need to stain or destain after running the gel) mix, heat at 65 C for 10 min or microwave secs, place on ice.

The third bit of information required is the voltage used for resolving your samples and do you circulate your buffer during the run. This is of particular concern when using TAE or TBE or any buffer system for which the pKA is > 7.5.

Without circulation at high voltages, a pH gradient develops across the gel. Since RNA is very susceptible to degradation at high pH, some of what you view on the gel may be a result of your running conditions and not the actual state of the RNA.

Lastly, you provided the A260/A280 ratio range.

Is the A260/A280 value you provide before or after DNAse treatment.

Since the untreated sample E lacks the HMW bands and complexes in the wells, then it is possible that you failed to remove the DNAse and the protein is stuck in the well. It would be valuable to know the A260/A230. That information is more relevant to whether you were able to remove contaminating substances from sample rather than anything that has to do with the picture you presented. It would also address the failure of your down stream reactions.

I would like to see a photo of your samples again, after removal of DNase and resolved under denaturation conditions with buffer recirculation, before I can be definite about the state of your RNA. Good luck

If you are sure all protein has been removed try adding [Final] ~60% formamide or ~4 M urea and heating to 65C for 5 min just prior to loading to denature everything. Works like a charm (see attached) What is the difference between the lanes? Why do some samples have well shifts and others not? This may be informative as to what the problem is.

Probably your sample are comtaminated with genomic DNA.

Your RNA looks to be intact from the image above . Lane D looks like there could be some degradation of the the sample . Is the RNA bacterial or eukaryotic ? Have the samples been DNAse treated. It could be genomic data contamination.

Do you have access to a Bioanlyser ? This gives you a better indication of the RNA quality by a RIN value .

Isolation of intact RNA is essential for many techniques used in gene expression analysis. Northern analysis, cDNA library construction and cDNA labeling for microarray analysis (especially when priming with oligo(dT)) require RNA of extremely high integrity. RT-PCR and ribonuclease protection assays both involve analysis of smaller regions of RNA (generally less than 1 kb), and, therefore, are more tolerant of partially degraded RNA. Regardless of the downstream application, it is a good idea to check RNA integrity before gene expression analysis.

The most common method used to assess the integrity of total RNA is to run an aliquot of the RNA sample on a denaturing agarose gel stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in native gels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species.

Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a very low molecular weight smear (Figure 1, lane 2). Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly (Figure 1, lane 1). Note: Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).

To check RNA integrity, there is an alternative method, e.g agarose gel analysis.

Currently there exists an alternative to traditional gel-based analysis that integrates the quantitation of RNA samples with quality assessment in one quick and simple assay. The Agilent 2100 Bioanalyzer (Agilent Technologies) is the first commercially available microfluidics instrument to provide detailed information about the condition of RNA samples. Used in coordination with the RNA 6000 LabChip® (a registered trademark of Caliper Technologies Corporation), as little as 1µl of 10 ng/µl is required per analysis. In addition to assessing RNA integrity, this automated system also provides a good estimate of RNA concentration and purity (i.e. rRNA contamination in mRNA preparations) in a sample. Previously, part of an RNA sample would be used for concentration and purity measurement (by A260 spectrophotometry) and another part of the sample would be used for integrity assessment. Using the LabChip® system however, concentration, integrity and purity are analyzed simultaneously in a single 5 ng sample. Data can be displayed as a gel-like image, an electropherogram , and in tabular format.

this could be a genomic DNA contamination...

Has the RNA treated for DNase? Try treating it with that and check again...

Its genomic DNA contamination. You can treat your RNA with DNAse, and the reprecipitate.

Hi all, thanks for your feedback. Sorry that i have forgotten to add the details. Sample lane A, B, C and D are DNase treated sample, while sample lane E is not treated with DNase. I have try to run Reverse transcriptase PCR for sample lane A to D for both RT and NRT, followed by normal PCR. No band seen in NRT samples. Therefore, i think it shouldn't be genomic DNA there. However, my another problem is the amound of cDNA generated through RT-PCR is very low as the band formed is normal PCR is very faint. I am not sure if this is something to do with the RNA integrity and therefore rise a question here..

Hi George Perdrizet, the different between lane is different concentration of compound used to treat the cell.

Hi Andrew Millard, it is yeast cell RNA, which is eukaryotic cell. My faculty do not have Bioanalyzer facilities and it can be only done by send service, which is quite costly.

To me it looks like only sample E is intact as the 25S-18S ratio is approximately 2:1. I think your DNAse treatment is causing problems. I'd recommend using Turbo DNAse.

I would also recommend purifying your RNA with Guanidium thiocyanate - Phenol solution and then do a couple of phenol-chloroform extractions. The guanidine thiocyanate will keep your RNA intact during the purification procedure. I can send you the protocol if you want. In my hands this gives far higher yields than any column based kit that I've tried.

Hi,

Your best sample is present lane E, the one not treated with DNAse, the other lanes are "OK" but not great (lane D degraded). One idea is that your Dnase prep has traces of RNAse contamination that could degrade your RNA samples. The DNAse treatment itself could contribute to degradation at an unknown step (incubation at 37°C or RT....). The material at the top of your lanes is probably yeast DNA.

Good luck !

I would suggest using Qiagen Kit for RNA extraction. It works well. The yield is good. The integrity of the samples can then be checked on 1% agarose gel prepared in 1XTBE and run at 200V for 20 mins. This works better than the 1XTAE. Regarding the other problem of cDNA being low, please check the primers first. You have mentioned that the normal PCR with genomic DNA is also faint. Hence I would check the primers first. Then try it with the genomic DNA first before proceeding. If that is fine, then I suggest you to use Fermentas cDNA kit for cDNA synthesis.

I would also suggest you to use QIAGEN RNA extraction kit, for quantity you can use nanodrop but for RNA integrity i would suggest you to use bioanalyzer and get RIN values for your sample if you don't have access to analyzer try using MOPS buffer for preperation of 2% agarose gel as well as for running. And make sure you are using DEPC treated/ RNase free water for same

hope this helps you

Well, if lanes A, B, C and D are DNAse-treated, this treatment didn't work. Lanes A, C and D clearly have gDNA contamination. I recommend that you check your enzyme system (the enzyme, buffer, etc. Are they contaminated?) and your protocol (are you using the correct proportion of DNAse/RNA?). Excessive amounts of DNAse may result in RNA degradation, while not enough DNAse may not be enough to digest all of the DNA contamination.

If the rRNA bands are of equal intensity, it suggests that some degradation has already occurred. Smearing below the rRNA bands suggests that you have low- quality RNA. Higher-sized bands indicate that the RNA pool is contaminated with DNA.

yes you have gDNA!

Hi, Phenol-chloroform method is ok. I recommend that you use two precipitations with NaAc 0.3 M and ethanol 2.5 vol 2-3 hrs at 4 oC, this help you to eliminate the phenol that some times is the problem with RT-PCR. Also, use AMBION protocol for run the samples in desnaturalizant gel. The concentration of primer y temperature are very important in RT (In my case 42oC works RT).

You seem to have some RNA activity in your DnaseI, you may have to buy Dnase that have been specifically manufactured for QRT PCR, which is of higher quality,

Use TURBO DNA-free kit (Applied Biosystems) to minimize gDNA contamination. It works very well. You can check the RNA quality using formaldehyde agarose gel electrophoresis, following instructions in the data sheet that comes with TRIZol. For microarray studies, we further purify RNA using Qiagen's RNeasy column. We also check the 260/280 and 260/230 ratios on NanoDrop. If you have access to Experion, RNA quality can also be determined.

Tzu, I wish to verify the information you provided. Firstly, you have provided a photo of your TAE gel displaying your resolved RNA samples. Secondly you have stated your A260/A280 ratio as 1.9 -2.1 and thirdly that you extracted your RNA using the phenol/ChCl3 method. Your question is about the material in the wells and the integrity of your RNA.

A few additional bits of information are required. 1. which of the phenol/chloroform methods did you use? Did you use the acidic phenol/CHCl3 method? (this is relevant to whether the high molecular weight in your lanes are genomic DNA or aggregated RNA.

This brings me to the 2nd information I require. Did you denature the RNA sample prior to running on the TAE gel.

I have resolved RNA on agarose gels with different buffer systems including TAE. You do not have to resolve your samples on formaldehyde or other denaturation gels for good resolution. I have obtained resolution on nondenaturation gels equivalent to that obtained with denaturation formaldehyde gels as long as I prepare the RNA samples in denaturation conditions.

(final concentrations: 50% formamide, 3.7 % formaldehyde, 1X (running buffer ,in your case 1X TAE) 1X dye solution, I also include EtBr to a final concentration of 10ug/ml, therefore I do not need to stain or destain after running the gel) mix, heat at 65 C for 10 min or microwave secs, place on ice.

The third bit of information required is the voltage used for resolving your samples and do you circulate your buffer during the run. This is of particular concern when using TAE or TBE or any buffer system for which the pKA is > 7.5.

Without circulation at high voltages, a pH gradient develops across the gel. Since RNA is very susceptible to degradation at high pH, some of what you view on the gel may be a result of your running conditions and not the actual state of the RNA.

Lastly, you provided the A260/A280 ratio range.

Is the A260/A280 value you provide before or after DNAse treatment.

Since the untreated sample E lacks the HMW bands and complexes in the wells, then it is possible that you failed to remove the DNAse and the protein is stuck in the well. It would be valuable to know the A260/A230. That information is more relevant to whether you were able to remove contaminating substances from sample rather than anything that has to do with the picture you presented. It would also address the failure of your down stream reactions.

I would like to see a photo of your samples again, after removal of DNase and resolved under denaturation conditions with buffer recirculation, before I can be definite about the state of your RNA. Good luck

Tzu:

Just a clarification. From the original post, I do not see any mention of DNAse treatment of the samples. Am I missing a part of the original posting?

Fedora:

A clarification: You mention that it is not necessary to resolve the samples on formaldehyde or other denaturation gels for good resolution. Yet, you resuspend your samples in 50% formamide and 3.7 % formaldehydeformaldehyde. Please explain how does this help resolve RNA.

In my lab we have been working at reducing the amount of noxious chemicals that are used. We tested the resolution of RNA under different conditions with and without denaturation of the sample prior to loading on the gel.

Denaturation gels such as formaldehyde gels were originally used to maintain the denaturation conditions throughout the run so as to ensure accuracy in determination of transcript sizes and efficient northern transfer. We have observed that it is not necessary to have formaldehyde in the gel but it is necessary to denature the sample prior to the run in order to achieve the level of resolution needed to avoid the types of secondary structure and aggregation that could give rise to ambiguous results on viewing the "resolved" samples. The formaldehyde and formamide RNA sample buffer with heating has worked very well to give well resolved RNA in agarose gels minus any denaturation solution or plus formaldehyde. Once the RNA sample has been treated for denaturation, the RNA appears to remain denatured even when resolved on gel systems composed of TAE, MOPS or in our preferred buffer system HE pH 7.2 (20 mMHepes , 1 mM EDTA) minus any denaturation chemical.

Thanks very much for your detailed response. I gather that continuous presence of formaldehyde is no longer necessary once the RNA has been denatured with formaldehyde. I am wondering how do you store formaldehyde treated RNA prior to analysis on a gel and how long can it be stored. This will be helpful for practical reasons.

I actually store my RNA long term in 100 % formamide and dilute or precipitate and resuspend in DEPC H2O as needed prior to down stream reactions. In fact in 100 % formamide we can store at 5C and maintain integrity . I only add formaldehyde just prior to gel analysis. Once denatured at 65 C, it is then placed on ice. I guess you can place it at -20 or 5 overnight until you are ready to load your gel.

Hi Tzu, for me, your sample (E) is perfect ( rRNA bands of equal size, no genomic DNA contamination, no degradation). Sample A can be used, if you don't care about genomic DNA ( high molecular weight band staying in the well). The rest of the samples are obviously degraded: 28 S RNA is weaker, than 18S, and there is also a smear below 18S. This tells you, that you have (or had) RNAse in your sample, and your polyA+ RNA , which you don#t see in the gel, is even more degraded. Think, what have you done differently for the sample E?

Sorry- correction: rRNA bands of equal intensity in the sample E.

Hi all, thanks for your all feedback.

Hi Prof. Fedora Sutton, i am using hot acidic phenol/chloforom for the extraction of RNA as suggested in YEAST Current Protocol in Molecular Biology and i do understand that RNA is prone to be extracted in acidic phenol. For the TAE gel, i didn't included any denaturing reagent such as formaldehyde or formamide. It is just plain 1XTAE and EtBr as pre-stain. However, i did included formaldehyde and formamide in RNA loading dye, which will be mixed with my RNA samples prior gel electrophoresis as stated in QIAGEN RNeasy Kit protocol. This method is suggested by my lab senior and do give them good indication of RNA integrity. For the voltage, the gel has been run for 75V for 40 minutes. Lastly, the A260/280 ratio were obtained prior DNase treatment, however, i couldnt provide you the A260/280 ratio after DNase treatment as now all the RNA have been converted to cDNA.

Hi, Dr. Sundararajan Jayaraman. i forget to include the DNase treatment info in the original post, but i do added the additional information as reply.

Hi Dr. Daria Onichtchouk. For the DNase treatment, actually i am using AMBION TURBO DNase for the treatment purpose, however, the packaging i purchased, did not include any DNase Inhibitor along, that is why i have to inactivate the DNase by heating the mixture of DNase-RNA with the presence of 15 mM EDTA at 75C. I am wondering will this actually affect the integrity of RNA and also the effeciency of RT-PCR? I already discussed this with my supervisor and we think it is better for us to get another set of DNase (Ambion DNA-free) which come with DNase Inhibitor and do hope this will actually help to maintain my RNA sample's integrity and remove gDNA

Last and but least, thanks again for advises provided.

Dear Hi,

"i have to inactivate the DNase by heating the mixture of DNase-RNA with the presence of 15 mM EDTA at 75C"

I would never ever use this procedure with my mRNA, this is not necessary and will only cause the RNA degradation!

1) you don't need to destroy the DNAse by heating, if you are doing phenol/chloroform extraction after DNAse treament step. Phenol/chlorophorm will remove DNAse completely.

2) how you are getting rid of 15mM EDTA,which will inhibit downstream RT reaction?

3) General and the most important question: what is the reason to use Phen/Chl, and NOT to use RNAeasy Qiagen kit?

This kit will give you superior standard results compared to ancient phenol/chlorophorm method. All you need is just follow the protocoll for yeast RNA, including on-column DNAse digestion option. We are routinely using RNAeasy kit in the lab, and are pretty happy with the results.

Hopefully it helps, good luck!

Actually the Turbo DNA-free kit from Ambion comes with DNAse inactivation reagent. This works well in our hands. Also, phenol/chloroform extraction method works very well. Although we find that the RNAeasy Qiagen kit is dependable, it is way too expensive. We use this kit for further purification for microarray studies. For regular gene expression studies, RT-qPCR, we do not have problems in using phenol:chloroform extracted and Turbo DNAse treated samples.

We get excellent m RNA using Trizol Reagent Invitrogen. Why not go for that. You just need trizol reagen, 100% chloroform for phase seperation, 100% Isopropanol for precipitation and 75% ethanol in DEPC treated water for washing and then finally dissolve in RNase free water or DEPC water. Its economical and gives good results.

I recently tried Bleach gel (see the link) to check RNA integrity prior processing sample for RNA seq. It worked good for me, please find attached the gel pic ( oops!!! the 1KB ladder is not great).  In the original article they used common household bleach with 6% sodium hypochlorite in the gel. I used clorax bleach with 8.25% sodium hypochlorite.  I made my 1XTAE from 50X and did not  denature RNA before loading, just added loading dye. 

 http://www.ncbi.nlm.nih.gov/pubmed/22222980

sorry was not sure whether you could see the link for Bleach gel article in the previous post

pKa of Tris is around 8.1, which means that Tris buffers in the low 7's (of which there are many) are greatly reduced in their buffering capacity (one log for each pH unit).

That means, Tris electrophoresis buffers, when below pH8 are not as stable.

and with RNA one has to worry about the pH going too alkaline leads to degradation problems. MOPS, with a pKa of 7.2, which means its buffering capacity is below pH7.

RNA is a single-stranded nucleic acid and tends to form secondary structures, a standard agarose gel will not give an accurate size separation of your total RNA or mRNA sample.Hence MOPS denaturing system is used.

I am having a hard time interpreting the integrity of the samples I ran on this gel. The bands are there but very pale. The ladder was a 1 kb DNA ladder since I did not have an RNA ladder on hand. Can anyone tell me what's going on with these samples and if they are degraded? Thanks.