Dear Mr. Azaz Khan,

1. Not clear with rna source- if trizol then too much tRNA remains in it. So random hexamer can give Cdna yield in which overall conc. Of you gene cDNA is less. My suggestion is to use oligo DT.

2. optimize the RNA amount (500ng to 1ug). Too much ot too less RNA lead to erroneous result.

Optimize on these two parameters –

1. template RNA (1ug at max)

2. Use oligo dT in place of Randox hexamer

3. Check for small length amplification (like 150-200bp). If it is +ve then clearly your RT enzyme is failing in long range cDNA synthesis. In that case with the above two optimization, you must achieve better results.

4. I feel your RNA yield is comparatively low (166ng/uL) and you need 8-10uL for reaction set up!!..

5. And what is your 260/280 ratio for RNA yield? Must be between 1.8-2.1.

Did you check your house keeping gene (GAPDH for example) in parallel? this will tell about the actual synthesis of cDNA with the method you are using. It is always advisable to consider the amount of template used. please check.

Hi Azaz

I agreee with the above comments.....you have to carefully find out what may have gone wrong.....it can be incomplete inactivation of DNAse (which can degrade your primers during RT) or even inefficient cDNA synthesis...give the precise conditions that your have used....if your using randome hexamers...incubation at room temperature is advised for primer annealing prior to raising the tem to $2 deg or so for RT ..as the primers are very small.....so check each step carefully...and as advised above first see signs of the reference gene you are using..

bye

Dear Mr. Azaz Khan,

1. Not clear with rna source- if trizol then too much tRNA remains in it. So random hexamer can give Cdna yield in which overall conc. Of you gene cDNA is less. My suggestion is to use oligo DT.

2. optimize the RNA amount (500ng to 1ug). Too much ot too less RNA lead to erroneous result.

Optimize on these two parameters –

1. template RNA (1ug at max)

2. Use oligo dT in place of Randox hexamer

3. Check for small length amplification (like 150-200bp). If it is +ve then clearly your RT enzyme is failing in long range cDNA synthesis. In that case with the above two optimization, you must achieve better results.

4. I feel your RNA yield is comparatively low (166ng/uL) and you need 8-10uL for reaction set up!!..

5. And what is your 260/280 ratio for RNA yield? Must be between 1.8-2.1.

I agree with all comments above and would like to draw your attention to Simanti's one. My first impression was that you might be using too much RNA in your RT reactions. I used to do this because the kit I had been using recommended a range of 500ng-1ug per reaction and always went for the highest amount. It turns out that the reaction might be saturated and therefore there is lower efficiency. Do a titration of the amount of RNA you use (500ng-800ng will be alright, as well) and then look for GAPDH expression in qPCR (you should expect a Ct of around 18-22).

Oligo DTs are safer because usually RNAs end in a polyA tail and therefore you will get global RT of all transcripts. I suppose that you nanodropped your samples to see how much RNA you have and assess its quality as Simanti describes above.

However, I disagree that amplification of 150-200bp products is a sign of failure; my products are always 100-150bp long and therefore I use the melting curve (SybrGreen technology) to confirm specificity of amplification. That is of course if you designed primers for such lengths of products. If you were expecting bigger sizes and you only get the small ones, then I agree with Simanti. To tackle this, use gDNA and repeat to get the products you expect (which is I think sth you did, right?). If you get the expected product size, then sth is wrong with the RT alone.

Good luck!

Using just oligo [dT]n's will give you 3' bias though - so, in order to get more 'global representation' with less 3' bias, a mixture of oligo [dT]n's and randomers (in a certain ratio; 1:5 for instance/BIO-RAD) is also advised. But, if using just oligo [dT]n's, try to design primers near the 3' end then (since that will be the region most voraciously/fully reverse-transcribed). ..

Other than that, I agree with the major point of the folks above who have stated that too much RNA can inhibit/confound the SSIII enzyme. Adding too much RNA to an RT rxn is like throwing too much wood on a good fire... it gets smothered... (plus, any RT-inhibitory crap in the RNA will alter SSIII's activity, despite product literature to the contrary; as there are many more sample types ("n") than there are kinds of SSIII enzyme: n = 1)

I.e., inhibitory agents in your RNA isolates (in addition to the RNA itself) can also wage war on reverse transcriptase activity. Different RT and 'Taq' enzymes are differentially susceptible to different inhibitors (see the excellent papers by Tania Nolan and Stephen Bustin on this debacle).

Also be sure your RNAs are of high quaility (e.g. Bioanalyzer RIN values of at least 5 and above [although others say 7 and above] and high purity before reverse transcription). RT-qPCR still fares pretty well even with moderately-degraded RNA since the relatively small regions of amplification are (by the law of averages) still largely intact, for the most part (which makes it a fairly stealth forensic tool on account thereof). But you don't want to get carried away with that thought too far... the 'law of diminishing returns' sets in at some point, as one might reasonably expect.

I agree with all above but did you check the quality of your RNA by looking the OD ratio (260/280). It is very important that you keep this between 1.8 and 2.2 for good quality RNA. Try another kit or a new your enzyme might be degraded.

Thanks to all of you for your constructive comments. Many of you are asked for RNA ratio at 260/280 and that is 2.14.

You guys are right I should bring down the amount of RNA, though checking DNAse if that is turn off is another good idea. I will go for another trial regarding your suggestion and hopefully I get something. Thanks

Frequent problem in reverse genetics is contamination of water. It's often ignore but can keep you in the lab for one year until you change the water or reaction mixture buffer. This works when quality of RNA is fine.

Also air... we get background RSV signal in all reactions during RSV season - twice a year ... like clock-work. Almost unavoidable -- with students, over-worked/worried professors, researchers and teachers sneezing in the building. Changing the water doesn't even help. Poor janitors!!!

Funny thing. Sounds funny - but is actually frustrating. Dust is everywhere, so always run NTCs. Naming the components of 'dust' is a worthy endeavor, t'would seem...

The A260/A280 ratio is not an indictation of quality - it is an indicator of purity. (It generally, in the absence of other things that can absorb at 260nm and 280 nm, tells one how much protein contamination is present in the RNA isolate). The A260 reading is of course (through Beer'sLaw; A = ebc) an indicator of quantity.

A better reading of quality would be obtained from a machine like the Bioanalyzer 2100 (Agilent) where one gets a spectrogram that shows how intact the RNA is compared to internal/appropriate kit standards. It also assigns an RIN (RNA Integrity Number) value to each RNA sample. You can have an A260/A280 ratio of 2.14 but have an RIN number of 3 -- indicating that the RNA is very pure, but highly degraded... (low quality)... so spectrophotometer and NanoDrop 260/280 ratios don't tell much about RNA quality. RIN numbers of 8 and above (10 being the highest RIN value) are desirable.

Thanks Jack for very useful information about RNA quality

Azaz,

Greetings to you. And I am glad that you found some of my comments helpful.

Azaz is just a great name - I wish my name was Azaz! ;]

Thanks! well Jack Gallup seems more sweet than mine, I dont know how you will pronounce my name as most of people struggle to pronounce it. Anyway you and all those who comments regarding my RT problem are valuable to me and I really appreciate it, I wish I get bands for RT today..finger cross

What did you find?

Hi Jack thanks for asking and I am sorry to be late to put my answer, actually I was away for some reasons.

Well. I bring down the Concentration of RNA 1ug/20ul Reaction and changed the protocol according to the manufactured Invitrogen Reverse Transcriptase III. I got very beautiful bands with Clathrin primer and with sox1 primer(my target one) but for the sox1 primer has a band of not correct size i was expecting (sox1 is used designed to used with DMSO buffer but I didnt used DMSO buffer in RT- PCR) so then I tried to used sox1 primer with DMSO but then there is no band as previously. I am still finding the answer is there something wrong with DMSO though I used a fresh stock of DMSO. It looks like my RT is working but problem is with Sox1 gene detection.

I have attached a gel picture which is of the RT-PCR without DMSO buffer you can see the inappropriate band of sox1 I am sorry if I havent explained it properly you can ask me again if you dont understand it I will try to explain it in a better way. Thanks

Use of DMSO can reduce certain Taq activity up to 50% and reduce secondary structure of G-C rich templates - but may not help in certain situations. Betaine can stabilize primer-template duplexes in AT-rich regions - perhaps Betaine (and not DMSO) would be worth a try. Use at ~1.2 M final concentration. You can purchase a 5 M solution of betaine from Sigma-Aldrich (cat. no. B-0300 ). It may be worth a try as it can improve product specificity... although it, like DMSO, can reduce Taq activity as well. But, to get your specific product, lessening Taq's activity is sometimes the right thing to do. Too much Mg++ can also be a problem, don't use above 3 mM Mg++ in your final PCR reactions to start with. Mg++ increases Taq activity - but at the expense of specificity.

Thanks for your valauble suggestions. I will definitely think about betaine and will let know about any improvement