You can make sure the quality of the RNA you are getting at first !!!!. Then secondly if you are using mammalian cells or tissues as source of RNA then u can try oligo dT primer once for cDNA synthesis. Later make sure the primer you are using are designed for the cDNA and not for the normal DNA else you will get errorneus result.

Best of Luck !!!!

For cDNA synthesis I can suggest you to mix the component in 2 step. First add RNA, Primer and water then put in 70 C for 5 min to be sure that all secondary structures are opened. After that keep the mixture in 25 C for 5 min and During this time you should add RT-Buffer, dNTPs, Ribo Block and enzyme. Then keep it in 42 C for 1 h and ultimately in 70 C 5 min for deactivating the enzyme.

Good Luck...

Hi Sagar. What is the quality of your RNA like? Do you have any contamination (230nm etc). Can you be more specific about the data - what do your reference genes look like - what are the CT values like?

hey,

I use the same protocol as Amir (small change: instead of 25°C I use 37°C), but in addition I also check before the RNA amount and do not use more then 500ng in the cDNA reaction.

if you measure the RNA and you use everywhere the same amount of RNA for cDNA synthesis your housekeeping gene should be detected at the same time.

if not something is wrong with the cDNA or RNA.

if it is the same but the results are still questionable you should check your primers for qPCR.

do you get just one product or more?

have you designed your primers over exonjunctions?

all the best,

Sebastian

Thank you all !!

RNA have ratio of 260/280 = 1.98. and concentration ~100 ng/microlitre.

I am confused that the cDNA is proper or not. can you please suggest optimum protocol for cDNA synthesis. i have random hexamers (Fermantas)

RNA quality was fine. i think gDNA contamination in samples.

Please send the detailed protocol from your lab at [email protected]

hey,

you should do also use DNase for your RNA extraction -> no gDNA left.

have you tested your RNA already on a gel?

I would just do the following:

take 500ng/sample for cDNA synthesis (in the qPCR you just take 5ul per reaction)

add water (final volume 10ul)

add oligo dT (or random hexamers)

heat the sample up to 70°C (5minutes)

cool down the sample

add dNTPs and reverse transcriptase buffer

put samples onto 37°C (5 minutes)

put samples onto 42°C and add reverse transcriptase to your samples

keep samples for 1hour

heat samples again at 70°C to stop the reaction

In the end you have to dilute your samples 1:10.

you end up with a Volume of 200ul.

that should work.

if you need any help let me know.

all the best,

Sebastian

Helo Sagar,

You can get cDNA using any of the standard technique.I suggest Roche kit. You follow the instructions provided in the kit. Then optimize your annealing temperature and template concentration using gradient pcr. Once it is optimized,easily you can follow it or you can standardize the same with the help of the technical expert of your PCR machine.

Make sure your amplicon is small, no more than 200 bp for better results, no primer dimmers and so on...all standard things to consider before starting. Quantify RNA with something other than NanoDrop (this machine is a random number generator!), RT each sample containing same amount of RNA 0.25-0.5 ug total RNA (20 uL reaction), take 2 uL and 5 uL each sample and run your standard PCR program (time, annealing temp, etc) but reduce your cycles to 22-25 (no more and even less if your transcript is abundant, you may have to play around with this number). If you have some preliminary real time info, check what's the Ct where this transcripts comes up at...your cycles should be around that. Semi-Q is not difficult, but it needs some standarisation. Don't forget your HK genes!

Best of luck!

PS: your RNA quality is best checked on a gel...your 260/280 ratio only tells you the RNA you have is "free" of proteins, good purity based on 1.98, the concentration means not much for quality. The best you have is a 1.5% agarose gel (not necessary to run a fromamide gel), and check that the 28S rRNA band is approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact.

Random hexamers for semiQ is fine. Your taq pol, use any you are used to.

PS: Follow this if ur RNA sample is from eukaryote 1. Isolate your RNA using Trizol (commercial kit) 2. Treat your RNA using DNASe 3. Check your RNA in Gel.. Quantify using Nanospec. 4. Take 1 micro gram and 5. do cDNA conversion using AB RT kit with oligodT primer . For your semiquantitative or real time PCR, use 1 micro litre of ur cDNA. As a precaution check your primer initially using ur gDNA if it is working with DNA then it should work in ur cDNA. store cDNA sample in -80 always. Gud luck friend.

I used a Qiagen for RNA isolation from high GC bacteria. The yield was good. The link is as follows:

http://www.qiagen.com/Products/Catalog/Sample-Technologies/RNA-Sample-Technologies/Total-RNA/RNeasy-Mini-Kit#orderinginformation

For cDNA synthesis, the following kit can be used.

http://www.thermoscientificbio.com/reverse-transcription-rtpcr-rtqpcr/maxima-first-strand-cdna-synthesis-kit/

Hi,

Check this paper out (doi:10.1038/nprot.2008.73). I think it's very useful.

Cheers,

Marcelo

Please don't forget to use an RNase inhibitor like RNaseOUT from Invitrogen mentioned by Gerbren Jacobs in your cDNA synthesis reaction.

Dear Sagar,

The basic thing which i observed in ur protocol is the termination step which should be done at 70 oC for 10 mins, where you are doing it at 60 oC. Once try with the change. If you are getting the same results, then let me know about ur complete protocol.

Thank you!

Hi Sagar

You have already got so many suggestions and I am sure these will be useful for your experiment....Iam also suggesting you some small but important points for your semiquantitative PCR analysis.... As many suggested it begins with a good quality RNA...and once you have made cDNA...you semiquantitative (or qPCR) analysis begins

1) always dilute your cDNA for subsequent PCR analysis......it is not only for reducing thr amount of cDNA (for clean amplification) ...but make sure you take more volume of cDNA soln. (to reduce pipetting errors).....e.g., instead of taking 1.0 µl (for 100 ng cDNA)...it is better to dilute it to 20 ng/µl and take 5.o µl as template.....errors at lower volumes will be eliminated

2) If doing many samples...make a master mix..

3) Also dilute the primers to avoid the errors due to low volume pipetting

4) The actual semiquantitative-PCR reaction has to be done appropriately...definitley it will involve optimization of PCR conditions like you do it for a regular PCR.....but for seeing the difference in expression of a gene you'll have to identify the 'cycle no' where the product is still not saturated...you'll have to try this for each gene separatly by removing the PCR tubes at 20 cyc, 23 cyc, 26cyc 29 cyc etc and see at which cycle number the difference can be seen.......it is like manually doing a real-time PCR where you can see the product amount after each cycle....but we analyze the Ct values and not the final cyacles when the products reach saturation and when you load them on the gel no differeence is seen........semiquantitative is more time consuming....but if there is no way out one has to do it...

5) use a reference gene also and normalize your gene of interest with the reference gene using a gel analysis software

5) try to use a qPCR instrument..if available in a neighbouring lab

bye

Below article providesvrequired info,

http://stanxterm.aecom.yu.edu/wiki/index.php?page=Semi_-_quantitative_RTPCR

Dear Sagar, as you have pointed out the critical steps in getting reproducible RT-PCR results are as follows-

1-Total RNA extraction and purification- give importance to the sampling process and initial RNA preparation steps. Grind your tissue samples in liquid nitrogen and always use RNAse away before start of the process. Should get reliable readings with Nanodrop Spectrophotometer. Store total RNA at -70C.

2- cDNA construction- Please ensure the quality and consistency of Oligo-dT primers. Sometimes it doesn't work. Use a RT-Premix. Store cDNA at -20C, but not for long. It is greatly unstable. I hope you can consider taking 2-4 microgram of template.

3-RT-PCR- Design the gene-specific primers well. Dilute cDNA (1:10) and primers to 10pm from 100pm before the PCR. I have seen that sometimes cDNA is effective without dilution as well, after my RNAi experiments. Do a gradient PCR to optimize the annealing temperature for the gene specific primers.

I hope soon you will be getting good results.

Try to not be confused because all of these suggestions, troubleshoot by the priority, ex; ensure your RNA is not degraded (I would suggest you to use the bioanalyzer), this is the main point, other suggestions will help you to get improved results. Best

As I see, you did not care about the integrity of the extracted RNA.

Please do it by simply running on the 1% agarose gel. To get better idea, use the specific gel for RNA which contains Formalehyde.

If you get the nice bands for the ribosomal RNA (the upper band has to have a double thickness), you are allowed to proceed to cDNA synthesis. You may have DNA instead of RNA, thus remove the suspicions by running on the gel.

Dear All,

As mentioned above comments I have followed the suggestions but still not getting proper results.

Please have look on my experiment.

Cell were treated Untreated, 50, 500 ng/ml LPS to mouse micrglial cells.

RNA was isolated with trizol. RNA integrity was also checked on agarose gel and it wad fine. Cancentration was determines by nanospect and 260/280 ratio was also fine.

One microgram of RNA was treated with DNAse I for 30 min (as per manufacturer's instruction)

one microgram RNA was taken for cDNA synthesis by Hicapacity cDNA synthesis kit (Invitrogen)

PCR:

2 microlitre of cDNA was taken as template.

PCR was done with 2X mastermix , 10pm primers (beta actin and TLR-4)

protocol:

95-10 min, 95-45sec, 60- 1 min, 72-1 min (27 cycles) 72-10 min,

Please find the attached Gel picture.

As per the reports TLR4 expression has to be upregulated dose dependent but it =s not happening.

Please help me.

Hi Sagar

You have already getting so many suggestions and I sure these will be useful for your experiments. all suggestion are useful for your experiments.

After RNA isolation from different samples take equal amount of RNA to make cDNA for different samples and then dilute the cDNA randomly like 1:5, 1:4 and first normalize your internal control (GAPDH, b-actin etc) then go for gene specific quantification and always make at least three biological replicate for each samples.

Hi Wilco,

I have done PCR using half template than previous and also reduced cycles.Please See the belove gel picture and suggest some modifications.

Why I am not getting dose dependent increasing expression?

i have used 23 cycles and 1 microlitre cDNA.

I think wilco is right

Have you simply tried another gene target ? I am not convinced that TLL4 will be increased. iNOS, or COX2 would be increased by many 1000's fold. Attached are two papers, one with info on TLR4 in BV2 and primary microglia. And another looking at gene expression after LPS.

And some genes that you could also try.

Article Characterization of phenotype markers and neuronotoxic poten...

Dear Dr. Bobbi,

Thank you so much, finaly I got the expected pattern for iNOS2 mRNA expression.

Can anyone tell me that what is the basic principle of 28s/18s ration in determining the RNA quality/RNA integrity? this ratio uses rRNA but will it work for mRNA integrity study? Is there any difference?

The ratio of 28s: 18s should be 2:1 it indicates the mRNA integrity also. if the ratio falls down it indicates RNA degradation in the sample.

thanks

Thank you very much Sagar. Can you tell me how to measure the ration from the gel? usually the 28s band should be double than the 18s, right? but how to measure the ratio?

Yes, the ratio should be 2:1. It can be analysed visually as the 28s bands intesity is two fold than 18s. If you want to know more you can go for densitometry analysis using Image J software.

thanks Sargar.

Hi,

Have you ever tried to purify your RNA, after TRIZOL extraction?

I highly recommend you to purify your RNA using some commercial kit, such as DNA-Free RNA Kit (Zymo).

This is because after TRIZOL purification it is very easy to have DNA contamination. This contamination can vary a lot, which would explain why you even following what our colleagues have suggested still don't have reproducible results.

If somebody have suggested that already I'm sorry about that - I only read some answers. ;-)

Best wishes,

@lex

Use the same number of cells for RNA isolation and take equal amount of RNA for different cells for cDNA synthesis and then randomly dilute the cDNA such as 1:3 or more according to your house keeping intensity then go for quantification either by real time PCR or other method.

Alexander it true, after RNA isolation treat RNA with Dnase enzyme to remove contamination from isolated RNA then go for cDNA synthesis

Treating RNA with DNase always helps, you could also try conditions 25 C for 10 min, 50-55 C for 40 mins, 80 C for 5 mins for reverse transcription reaction.

First of all please check the abundance status of the gene you are interested in. If its a low abundant gene, please check your RNA isolation protocol for its efficiency for low abundant mRNAs.

Secondly, check the quality and quantity of your RNA.

DNase treated RNA should also be checked before proceeding towards RT. (The ratio of 28s: 18s should be 2:1 it indicates the mRNA integrity, already suggested by other coworkers as above).

The RT enzyme you are using whether have RNase H activity or is required separately. (also in RTs which have RNase H activity, additional RNase H is required in case of genes more than 1 Kb).

These are few things I have experienced in research work.

All the best!!!!!

You could try using one-step RT-PCR, there is less error. There are plenty of one step reaction mixes out there - just add your total RNA and gene-specific primer pair. It does the reverse transcription from the gene-specific reverse primer then switches straight to cycling in the same tube. Chuck in the same amount of RNA to each reaction and you can do controls with GAPDH or PPIA or whatever. Once you have figured out how many cycles to do its very consistent.

Because the reverse-transcription step is gene-specific, you don't need use much total RNA in each reaction... so its very efficient.

Use primer 3 (online, free) to help design the best primer pairs, it makes a huge difference in efficiency. Keep the products short - it works better and saves time (and causes less problems if your RNA is a bit degraded ). Try to design primers across exon-exon boundries if you can.

In addition to treating your RNA with DNaseI, you should control for DNA contamination by showing that conventional PCR fails to amplify any products from your RNA sample. You can also add RNaseA (careful!) to show the bands are RNA-specific.

I have a question. Is it necessary to use molecular grade ethanol when we add to the wash buffers in the commercial RNA extraction Kits? or we can use analytical or HPLC grade one. does it really affects the RNA quality?

We use the Sigma grade or atleast pure grade of ethanol. But i have not seen performance dramatically change with analytical grade ethanol as well.

Dear Bharat, do you think then HPLC grade should be ok?

I mean is that analytical grade chemicals do work fine and you can get reproducible results. But should not put it into practice and once molecular biology agents are available, please use the same. HPLC grade reagents are designed for specific purposes and are costly, and are extra fine, it may not be cost-efficient to use the same for molecular biology routine labs.

Sagar, the best method is that u can dilute the cDNA and make sure that ur primers have a Tm of 60degree and amplicon size less than 150bp.

First perform reverse transcriptase PCR and confirm that there is no dimers then finally go to QPCR.

The dilution u can make 1 in 5 , 1in 50 and also directly go for neat sample.

the cDNA quantity u used was good enough. Try to use POWER SYBR green(ABI) or FAST SYBR green (ROCHE).

Check the sybrgreen compatability with ur machine specification.

Hope this will work

Cheers

I actually have one question... Why are you using Nanospect?

You could use other quite well established methods/software for the semi-quantitative analysis.

HI, I do agree with everyone's suggestions above.

In my opinion, however, you need to start with contaminant-free RNA template. we also use TRIZOL method for some of our samples but we have also experienced problems moving directly from this to QPCR with no consistent results. I think the alcohol washes are not sufficient to remore the solvents from the first steps.

To solve this I have optimised the protocol by adding another step doing a lithium cloride precipitation and ethanol washes, the resuspend in RNAse -free water.

Alternatively, after the isopropanol precipitation and wash the eluted RNA is re-purified using a commercially available kit i.e. qiagen which also contain a DNAse step to get rid off genomic DNA. Our qpcr results have improved dramatically after that.

for QPCR you need to make sure you don't have primer dimers formed or tertiary structures and ideal Tm of 60 so you can run multiple genes on the same plate if needed. Make sure the amplicons are between 100bp and maximum 200bp long.

we use the nanodrop for ODing the samples which gives you the 260/280 ratio which should be 1.8-2.1 for best quality.

For starting material i tend to go for max 500ng from each samples for cDNA. I then dilute the sample down to 5ng/µl and then use equivalent of 10-20ng per qpcr reaction. We tend to use SYBGreen for most of our primers which you can get from a few companies but check compatibility of product with your machine.

Also always use a house keeping gene (i.e. gapdh, beta-actin) to run at the same time as your control.

if you are not happy with just the qpcr machine results you can always run your samples on a gel to double check.

I hope that helps.

Good luck

Hi, I agree with some good advice by Penelope for qPCR analysis..

I used for RNA extraction SV total isolation system from Promega. and Q-a-gene PCR kit. BUT, I used to do standard curve. it gives trustworthy results.

Now the new qPCR is the ddPCR (droplet PCR), which not require standard curve.

also I suggesting use pico determination amount of RNA to be constant using RNA template. using Agilent RNA 6000 Pico kit.

Your qPCR data is as good as the quality of your reagents as previous contributors have eluted to already. However, make sure that the efficiency of your primers is between 95 and 110 % and that you get a single melting curve when using sybr green. Also, the choice of your reference genes to be used in your system is critical. Anybody doing qPCR or ddPCR should adhere to the MIQE guidelines in order o make your data reproducible!!

Go for fermentas cdna synthesis kit

You haven't mentioned DNase1 - however, Ms Tsimbouri has in her answer. There are many bells and whistles that can be used - I used to do a lot of real-time RT-PCR from bovine and mouse embryos. The amount of RNA isolated does not seem to be limiting in your case, but the DNase1 step is crucial in RT-PCR. You might like to ensure that your primers are across the intron-exon boundaries, or are in different exons to ensure that genomic DNA is not amplified - or at least can be differentiated on a gel, real-time is a bit problematic, because the fluorescent signal will still be measured and contribute variably to your Ct, which could account for your inconsistent results. So...do a DNase1 step - make sure to denature it - can be a bit hard to kill - and run an RT negative (no reverse transcriptase) so you can check for genomic contamination. If your primers are not across boundaries or in the same exon, you cannot get by without a DNase1 step - your results are pretty meaningless - sorry to put it so bluntly...but that's the case!!

If I get the point correctly, you can make cDNA and run a standard curve by qPCR from serial dilutions of cDNA template. Then you can calculate the copy numbers of the gene of interest based on the standard curve. Sometimes this way is helpful for the cases that you have no reference gene. I have done it and is easy to perform, just need to be careful while you are making serial dilutions,,, Good luck