RT-PCR is a little complicated sometimes. What kind of RT are you using? Is the amplicon short or long? Have you designed so that you are between two exons (so that hnRNA will be EXCLUDED from the result?). Here are some in silico tools that you should use to check the sequence match (but you will want to look in dbSNP and be certain you haven't made primers that include known polymorphisms, especially at the 3' ends):

http://www.ncbi.nlm.nih.gov/projects/e-pcr/

http://genome.ucsc.edu/cgi-bin/hgPcr?command=start

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Assuming you have done everything correctly, and your primers are between 18 and 22 base pairs long, and have been correctly synthesized, your PCR conditions become suspect. The main thing that goes wrong in PCRs is bad nucleotides. These must be kept very cold and separate and are only good for certain periods of time. Write back with what you find. No question this can be repaired. Anything known can be PCRd to some extent.

How you desgned the primers? Do you have control the rapport among G-C? Do you have used the right annealing temperature? how many cycles?

Did you blast those primers? Did you do PCR for those primer before you did RT-PCR?

Can you give more details, such as sample amount and quality, RT reaction, and system you are using? Is your reference gene working well? I've come across problems trying to detect low level transcripts, which typically require a pre-amplification step that keeps a relative ratio between samples.

RT-PCR is a little complicated sometimes. What kind of RT are you using? Is the amplicon short or long? Have you designed so that you are between two exons (so that hnRNA will be EXCLUDED from the result?). Here are some in silico tools that you should use to check the sequence match (but you will want to look in dbSNP and be certain you haven't made primers that include known polymorphisms, especially at the 3' ends):

http://www.ncbi.nlm.nih.gov/projects/e-pcr/

http://genome.ucsc.edu/cgi-bin/hgPcr?command=start

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Assuming you have done everything correctly, and your primers are between 18 and 22 base pairs long, and have been correctly synthesized, your PCR conditions become suspect. The main thing that goes wrong in PCRs is bad nucleotides. These must be kept very cold and separate and are only good for certain periods of time. Write back with what you find. No question this can be repaired. Anything known can be PCRd to some extent.

As a physician, not trained as PhD, I prepare my primers using primer3 and blast them using Pubmed website. Did you obtain cDNA from mRNA appropriately? Was your RNA well prepared? How long is the expected size of product (150-200NT is better) by the designed primer set? That is a few things from a RT-PCR user.

The problem cannot be solely attributed to your primers. Check your RNA yield and quality immediately after extraction by at least running RNA gel. An observation of three distinct bands in an agarose gel electrophoresis will depict good quality, and the strength of the band is an indication of the yield. This check is very important owing to the fragility of RNA occasioned by the ubiquity of RNase enzyme. When you are sure of your RNA quality, then you can worry about other factors like your primers.

Best wishes!

I just wanted to write that I agree with Benedict Odii above that RNA quality is always an important factor for proper RT-PCR. Nowadays, since people often use total RNA extractions with kits like RNA easy, keeping RNA clean and avoiding degradation is nowhere near as difficult as when I started working with RNA in 1977. I assume that you are using high quality commercial kits or reagents to extract your RNA and that you keep your samples cold, work quickly, measure ODs and run gels to be certain of RNA integrity before you start your PCR work.

Hi everybody. The sequence I selected from transcriptome results corresponded to the gene BAX1, involved in apoptosis pathway. Does any one has experience with this? I checked in BLAST and it correspond to the same gene in other organism. I designed the primers in primer3 (frodo.wi.mit.edu), the amplicon length is between 97-127bp, primers are around 20bp, there is not more than 2ºC of different among them and not more than three C or G toghether. I have perfomed the typical PCR but I never obtain any amplification. Actually, I have tried different samples to be sure about the quality. My ARN has a ratio 260/280= 2 and 260/230= 1.8-1.9. As Bert says, we used the kit RNA easy in the lab. However, I do not see nothing after PCR so, could be my sample wrong?

Hi,

yes something is wrong. Not getting anything with any PCR suggests that the problem is not in the primer design.

This is a complex protocol, and from the limited information we are given it is not possible to pinpoint the mos probable mistake. You have not mentioned the reverse transcription, if it is done in one step with the PCR or before in a separate reaction, if you are using oligo-dT or specific primers or random hexamers, etc. If your RNA is not reverse transcribed properly, you do not have anything to amplify.

If you get some RNA (how much do you have?) from the isolation, which is free of genomic DNA and you use some reasonable reverse transcriptase (What are you using?) than you should have some cDNA. If you have it, then by decreasing the stringency (lowering the annealing temperature or increasing Mg concentration in the conventional PCR) should result in SOMETHING. At least byproducts from mispriming or primer dimers, if your PCR primers are of wrong design.

You probably loose you RNA or have some missing reagents or inactive enzymes somewhere. I would suggest you to step by step confirm the success of your protocol, The existence and quality of the RNA can be assessed on a gel, as written above. Try using random oligo-dT for RT (what RT primers were you using?) and use some control primer pair. For example you can design a primer pair that would amplify the ribosmal DNA from the genome. If it works form DNA extract, it should work on cDNA too. If you have amplified product using conventional enzymes or kits, you should be able to repeat the same using Q-PCR kit in the previous setup

When you locate the error using robust, less specific and non-quantitative reactions, you can change back to the delicate Q-PCR.

Do not forget that if there _IS_ a problem, and you were searching for it everywhere that you can imagine that it is at, and you have not found it, then it is somewhere where you would not imagine to find it. :)

as mentioned above, your problem is not mandatory linked to your primers. Quantification of your starting RNA by spectroscopy do not preclude an failure of your RT-PCR due to the fact of e.g. bad RNA quality/integrity, putative contamination with inhibitors or less or overdosed amounts of nucleotides. Intact and degradedRNA (e.g. by RNAses,...) show very similar UV absorbance as well as ratios when quantified in a spectrometer. Therefore, quantifiable detection of RNA is no adequate criterion for suitable RNA quality.

First, you should generate a positive control RT-PCR reaction, using well established primers to amplify a positive control gene (e.g. house keeping gene GAPDH,...). If you are succesful with this RT-PCR you can test different amouts/ratios of your single primers of interest. Sometimes, different amounts of forward and reverse primers are necessary for efficient amplification. In addition, a gradient of Mgcl2 (1-1.5-2-2.5) may be useful.

If primers and RNA-quality were checked, you may test your RT-reaction. Did you used random -, oligo-dt - or specific primers. Test to pre-incubate RNA and primers (without reverse transcriptase) together for 5 minutes at 65-70 C to neutralise secondary structures and to improve primer hybridisation, cool the mixture on ice and then add the enzyme. To improve primer binding start the RT -reaction with an incubation step of 5min 30 C then further increase the temperature to the optimal RT-temperature.

You may mix random and oligo-dt-primers to efficiently covering long transcripts/cDNAs.

so, it seems I should confirm the quality of the samples.

Peter, the quantity of ARN is highly variable. It is from hemocytes and the number of cells obtained from each animal is not always the same. However, the quantity was standardize to 1ug/ul previous to perform reverse transcription with Maxima First Strand cDNA kit (Thermo Scientific).

I've never used that kit made by Fermentas that appears to use Moloney Murine Leukemia Virus RT in combination with a random hexamer to give you a first strand synthesis. I do NOT have a .pdf of the product insert, with which I might be able to help troubleshoot. You are welcome to send a pdf to me at [email protected] Now, the biggest problem with first strand synthesis is (1) undegraded RNA (2) getting the concentrations right (3) making sure the buffer conditions are correct and (4) freshness of the rt enzyme. Once you get a good first strand, second strand and PCR is usually not a problem. Are you at 50oC for about half an hour for the reaction? Do you immediately dilute? Are you using a range of starting RNA concentrations? Do you have a control RNA? Answers to each of these questions will help you figure out what's going wrong.

Thanks a lot Bert. It sounds I will check all the things posted.

and I suppose I'll back with more questions. Thanks a lot everybody for the answers

Without going into details of the your primers not working. First check point is do you do a positive control alongside the test one in your RT prep? Does the positive control work? if that works and the test does not work then comes the question to deal your test ones?

Mary