Did you include a positive control for PCR (such as genomic DNA or something)? This tells you whether your primers are working or not. What kind of primer did you use for the 1st strand synthesis? Oligo(dT), random hexamer, or gene-specific primer? Each one has its own advantages to another, e.g. oligo(dT) might not be suitable if you are to amplify the 5' proximal region of a relatively long transcript. Did you optimize your thermal cycling? I've seen for some extremely rare transcripts that the reaction product starts to accumulate only after >35 cycles. If you mind increasing the number of cycles, you can increase the amount of cDNA in your PCR. Did you try using some PCR additives (Betaine or DMSO)? If the region to be amplified is unusually GC-rich, such additives might help to allow proper amplification. The RNA quality can be an issue as well. The success of PCR with one amplicon does not guarantee the quality of starting RNA, because some abundant transcripts (such as those for many housekeeping genes) tend to be well-amplified even though the starting RNA has been degraded to a certain degree. There can be 100 different reasons for the failure for 100 different reactions.

Are you looking for splice variants? Then It could simply be that the splice variants are not expressed or only expressed at very low levels. 

A positive control is always good to include. You could use genomic DNA as positive control as already suggested, if your primers are not exon-exon spanning or placed in different exons that a positioned 1000s of kb apart. You could instead attempt to make a positive control by cloning, but it may take a while if your transcripts are only expressed at low levels in your model system. 

Your issue could however also be related to primer-design. If you used an older reference sequence for primer-design, it could be that, what was once interpreted as an exon in reality is an intron. So be aware of what reference sequence was used for primer-design.

Cheers,

Tina

Here is my suggestions:

1. Check that you primers are working optimally. This can be done by testing them with  genomic DNA or cDNA (depend on the design). Also test all your primers optimal temperature.

2. Always use internal and positive control. For the positive control always best to use confirmed cDNA from WT sample for example. This important to make sure all your primers and PCR properties work properly. The internal control important to make sure that you use the comparable input of cDNA and more importantly to make sure that your cDNA synthesis worked.

With this two initial step then you will get some clue where the problem coming from.

1. If the primers not working, then you should do some optimization from the primer design or from the PCR design (i.e using G-C rich mix, different temperature, etc)

2. If the internal control don't appear you should optimize the cDNA synthesis part. Here you can play with temperature or the rt-primer you used.

3. If all control worked fine then it means your genes that didn't appear are simply not expressed.

Good luck!

You can always try to do a little bit of bioinformatics work before your lab experiments, to find out if the gene you're looking for is expressed in the rice tissues you're using. I am sure that there should be some rice transcriptome-expression data in the databases, where you can see if your gene is expressed (normally the input in these databases is the gene's ID). If it does regularly and abundantly, then you should follow the advice given by the colleagues above. 

I think you need to answer the following questions first. Have you checked the integrity of the extracted RNA? Have you used sequence specific primers in the cDNA step or other primers and how relevant are they to your transcripts? Have you optimized the primer design and the PCR conditions or not?

I actually got a clone encoding for rice lipase from rice genome resource centre. I am going to study the function of this gene. Therefore, I tried to clone this gene and express it in E.coli. All my three pairs of primer work on the plasmid clone. For my next part of my project, I planned to study the expression of this particular gene. Therefore, I extract the RNA and then synthesize the cdna. I use the same parameters on th cdna but they failed to work. As I mentioned ealier, one set of my primer  works. I have send them off for sequencing. There amplifies the correct region but the others two sets just doesn't work at all

If your primers work well with the plasmid clone, it means that they are okay. Also you have applied the same conditions. Thus the problem is with your extract. If only one primer works, it means that the reaction is free from inhibitors and the reagents are okay. Perhaps you dont have transcript for the other 2 primers or you have some sort of degradation that affected this particular region or genes. Are you using conventional RT-PCR or real time PCR?

I have repeated my rna extraction for abt 4 times and I get the same results everytime. I am doing end point pcr 

use 1/10 of your PCR as a target for a second PCR and check the results and use silver nitrate staining for visualization or syber green. Alternatively you could use Real time PCR to be sure that you do not have a tarnscript at all from those two genes.

Dear Shanice,

from your answers I understand the problem most likely comes from the RNA/cDNA, since the primers are working with a plasmid. Two ideas come to my mind:

1.- You could have directed one or both primers in a primer set to an intron.

2.- There might be an issue wih the kind of retrotranscription you're performing. I'd recomend you to use a universal mixture of oligo-d(T) and reandom hexamers for a better performance rather than specific primers. This days there are some comercial first strand synthesis master mixes for qPCR that already contains this kind of primer mixture.  Also, as pointed out by Dooyoung, it could be more difficult to amplify regions near the 5' in long genes so, unless required for your research, tend to set your primers near the 3'. This also helps when dealing with partially degraded RNAs, since degradation starts by the 5' end.

There are many other things to check too, if you're already checked for these things you'd need to provide more details on your actual methods to help on the troubleshouting.

Are your housekeeping gene primers working for the cDNA samples?