I suspect RNA degradation. To confirm or rule this out,run a small sample of RNA on an agarose gel, to see if you can observe sharp bands for the 18S and 28S rRNAs. 

your bands are very strong so I wonder if you have too much amplification and the product is self annealing and randomly getting longer as the product overtakes the primers in concentration in the reaction. How many cycles and how much starting material are you using. Have you often used actin before with these primers and could you have post pcr contamination from previous experiments? . Do you negative controls show an actin band ?

Dear Craig , RNA is enough good on agarose gel(2%).So i think there is no problem related to RNA .

Dear Paul Sir, I used 55 cycles , cDNA is same for all , the primers have been freshly reconstructed and then diluted (3pm), negative control deos not show  actin band 

I must say that 55 cycles seem very long to me. If your are going to measure quantification, it is not possible anymore. If your measuring yes - no, then its ok.

There is a reason why al qPCR programs standard do not go further then 40 cycles in there program (you can set higher if you want, but standard it is set to 40).

Above 30 cycles your reactionmix gets used up, (dntp's, etc.,) your polymerase is also degrading. So you will get off target results after that, even negative thing might become positive for some signal. Do 1 sample 30 times and you will have 30 very different results if cq is above 40, and you go to 55 cycles. If not, please show me the results because you would have acomplished something i have never seen in my career.

If your cq is above 40, especially for actin, you have a problem. Is actin stabaly and abundantly expressed in your sample? if not, its not the right housekeeping gene. and you will need to search for another one

The best values for housekeeping and unknown samples are between 15 and 25. You can go up to 35 for your unknown, but stretching to 40 and above introduces huge variations which render your analises useless.

Again this is for qPCR, if you would perform a regular PCR where you are looking at a yes / no answer, you can go above 40 cycles no problem

Thanks Dear David for ur informative comment.  But i m using same 55 cycles for transporter protein primers , why only actin shows the smear . May actin primer has any problem ?

Hi Sheikh, thx for respons, can you give me the Cq (quantitative cyle) for your actine values and for your GOI? Have you used this assay (transporter protein) already before? From what i can deduct from your initial question, is that you are working on plants, I have no experience on plant material and expression, so i am extrapolating my experience from mamalian and mouse material.

is your actin assay a recent designed or purchased assay, or was it already used in past? Do you know exact where your actin primers are located in the gene (exon/intron spanning) if your primers are not exonspanning, you might have off targets with genomic DNA.

Dear David for  Actin Cq value plz see   above image. And i m optimizing the primers for expression analysis (qPCR) first time, i m working on plant Coleus forskohlii

In the second picture above I see sample 14 at 31.65, but for the rest mainly meltcurve analisis. So i do not know if this is your bactin or GOI.

With cq of 31.65, there is no need to go to 55 cycles, just a waist of time ;)

in the gelimmage i see 1 smearlane, (wich is actine i presume), the rest give nice intense amplicons. Something else that just now got my attention, you have a 250bp product for bactin, for quantitative PCR this is quite large,, something between 50 and 150 to 200 is max. I try to have an amplicon of around 100bp for most of my assays.

How big is your experience in quantitative PCR? So i know what questions to ask and which i can leave out. Maybe you can provide me with more details regarding what you want to measure, what your end goal is. So i can help you more to the point.

Dear Sheikh

I totally agree with David, the problem may reside in your reference gene primer`s design and product size. Cq of 31.65 reflects nearly no amplification

Thanks, I will avoid these mistakes in further experiments

Hello Dear Friends,

Now i used a new  set of actin primer(180 bp) . I checked a normal PCR reaction with leaf cDNA  which showed clear  bands (at 55 and 57 Ta both) on 2% agarose gel(u can check from the image below). But when i did its qPCR no amplification was seen. Plz suggest a clear and helpful solution to my problem?

not realy much of ideas, unless you didn't use a sybr green mix for the qPCR. I hope you wherent using a regular mastermix with the qPCR. Check if all components where correctly added to the mix.

For gelimmage is this the reaction mix from your qPCR run, that you pipetted on gel? If yes, there is something wrong with your machine, or the mix didn't contain sybr green

Gel image is of  normal PCR  not qPCR. SYBR Green is OK . Is there any issue of primer annealing becoz in normal PCR i used annealing at 55 for 20 sec and in qPCR at 55 for 10 sec? If my Cq is high , how to reduce it so that optimal amplification can occur in even 40 cycles  ?

10 second annealing is very short

My regular program is more like this

95°C 10 minutes (initital denaturing)

40 cycles of 15 seconds denaturing  at 95°C, 30-60 seconds (depending if the enzyme is a fast or regular enzyme) annealing - elongation at 60°C (I optimise and design all my primers to work at 60°C) So all assays can run on almost 1 programme so running different assays in 1 plate is possible.

So In my opinion, your annealing should be a lot longer (at least the 20 seconds you use in regular PCR, recommend even 30 seconds and at least 30 seconds at 72°C for elongation if you used 72°C before. Your primers simply do not get enough time to find there math in the DNA pool, which results in no amplification

this is why it is easier when we have more information on how you work and which programs you use. We can find problems faster. When we do not have the info, we assume normal rules and assume you use quite standard techniues and programms and mixes. We will not steal your data away, most of us do not even use plants in there work en  simpply want to help a collegue out ;)

If you require more help, I 'll be around

grtz

David

Very thanks Bro David for ur kind and quick help . But i had no intension that somebody can steal my data. There is nothing like that.