I am having trouble in PCR to amplify a low abundant gene. cDNA is made from total RNA. I was always adding 2 ng/ul - 4 ng/ul cDNA in the PCR reaction. Positive control like GAPDH always shows up brightly. But my target gene never showed up. I've tried different annealing temperature, adjusting the concentration of Mg++.
Anyone can give me some suggestions on PCR amplifying low abundant gene?
Hi Yue
as mentioned by Christiane it isn't clear if you are performing gel based RT PCR or sybr green (real time) q PCR
1. If using cDNA for gel based RT PCR then PCR can work between an amount of RNA corresponding to 10ng to 100ng: If performing a 20ul cDNA synthesis reaction (diluted to 50ul) this typically corresponds to about 1-2ul of your resulting cDNA. REMEMBER: You CANNOT nanodrop your cDNA directly at 260nM because of unincorporated dNTPs (thus you massively overestimate apparent cDNA by Nanodrop without column clean up or ammonium acetate precipitation of your resulting cDNA reaction). Thus, you either make reference to your starting nanodrop RNA; clean up your cDNA by column or by ammonium acetate + ethanol ppt) or using fluorimetry (Qubit or Spectrophotometry with fluorescent dyes).
However, I am digressing: However you acertain cDNA conectration for gel based RT PCR 10ng-100ng is the usual permissive range so with new cDNA and/or primers I will routinely verify expression and/or whether the primers work well by adding 20ng to 50ng of cDNA to a standard PCR reaction
2. For real time (sybr green) qPCR the usual range is about 1/10 to 1/100 x that amount: or 0.2 - 5ng of cDNA (typically) - so what you are using
3. To go back to your original question for low copy number genes you should aim to amplify at least 1ug of total RNA in your RT reaction
4. Moreover, apart from a 260/280 ratio of 1.8-2.2 make sure your 260/230 ratio is > 1.0 or preferably > 1.5: For high copy number housekeeping genes, even with salt and in particularly GITC contaminated RNA (indicated by a 260/230 ratio < 1.0) you might have high enough copy number to subsequently detect your housekeeper but not a low copy number gene of interest
5. As you say, if you have made sure that the Ta of your gel based PCR or sybr green qPCR is between TM to Tm -5C and invariably Tm-2C and also increased your Mg in 0.5mM increments then you need to consider the placement of your primers; how you carry out your RT reaction and also potential secondary structure in your target:
Take a look at a prior answer of mine
https://www.researchgate.net/post/Should_I_use_random_primer_or_Oligo_dT_ror_relative_quantification_of_gene_expression_in_Q-PCR
5. In essence you need to make sure
- Your primers are within 1KB of the polyA tail if using Oligo dT in your RT reaction
- Better still use Random hexamers in your RT reaction; either with or instead of Oligo DT
- Make sure your amplicon is 100bp-200bp in length (not say 500bp) to maximise efficient qPCR or gel based PCR
- Select a region where predicted target secondary structure does not appear to exist using packages like m fold
http://unafold.rna.albany.edu/?q=mfold/mfold-references
- Similarly, screen your low copy number GOI primers for self or hetero dimers (primer dimers) to ensure efficient amplification with of your low copy number gene using something like IDT oligo tools
https://www.idtdna.com/calc/analyser
See my own word doc I designed for students to maximise (optimise) primer performance (attached)
6. Finally to conclude I would say having made cDNA from at least 1ug of high quality RNA (260/230 > 1.0 and thus devoid of GITC) preferably using random hexamers try amplification of small (~200bp) fragments either using standard PCR @ Tm-2C or as suggested by Giacomo by touchdown PCR, which I have got to work very well (and is especially effective if the Tm of your primers is > 2C)
Ive definitely said enough !!! Good luck !!
If you have access to cDNA for gene of interest in a plasmid, it may be helpful to optimize conditions for PCR (annealing temperature and concentration of Mg++ etc.). After making sure that the conditions are optimum, I would use these conditions and add increasing amounts (5-100 ng) of cDNA template and try different amplification cycles and analyze the PCR product on the gel. Using these systematic approaches, it should be possible to identify potential problems in the detection of low abundance mRNA in your experiment.
Hi Yue,
Before answering your question: Are you doing qPCR because you mention GAPDH as "control" is working, or are you doing normal amplification of your target gene with cDNA?
Dear Yue,
I have had a similar issue once and it worked out by performing a touchdown PCR followed by a nested PCR, even with the same primers.
First, try to add as much cDNA as you have in the first reaction (tot volume = 50 ul). Perform two cycles with a Tm 10ºC higher than that of your primers (or of the one primer with the lowest Tm), then two cycles with a Tm 5ºC higher, then 15 cycles with the exact Tm.
Once the reaction is over, take 5ul and put them in a second reaction to a total of 50 ul. Perform 35 cycles with the exact Tm and then run on gel.
If that doesn't work, you may want to try decreasing the concentration of the primers... sometimes that helps.
Best of luck.
Hi Yue
as mentioned by Christiane it isn't clear if you are performing gel based RT PCR or sybr green (real time) q PCR
1. If using cDNA for gel based RT PCR then PCR can work between an amount of RNA corresponding to 10ng to 100ng: If performing a 20ul cDNA synthesis reaction (diluted to 50ul) this typically corresponds to about 1-2ul of your resulting cDNA. REMEMBER: You CANNOT nanodrop your cDNA directly at 260nM because of unincorporated dNTPs (thus you massively overestimate apparent cDNA by Nanodrop without column clean up or ammonium acetate precipitation of your resulting cDNA reaction). Thus, you either make reference to your starting nanodrop RNA; clean up your cDNA by column or by ammonium acetate + ethanol ppt) or using fluorimetry (Qubit or Spectrophotometry with fluorescent dyes).
However, I am digressing: However you acertain cDNA conectration for gel based RT PCR 10ng-100ng is the usual permissive range so with new cDNA and/or primers I will routinely verify expression and/or whether the primers work well by adding 20ng to 50ng of cDNA to a standard PCR reaction
2. For real time (sybr green) qPCR the usual range is about 1/10 to 1/100 x that amount: or 0.2 - 5ng of cDNA (typically) - so what you are using
3. To go back to your original question for low copy number genes you should aim to amplify at least 1ug of total RNA in your RT reaction
4. Moreover, apart from a 260/280 ratio of 1.8-2.2 make sure your 260/230 ratio is > 1.0 or preferably > 1.5: For high copy number housekeeping genes, even with salt and in particularly GITC contaminated RNA (indicated by a 260/230 ratio < 1.0) you might have high enough copy number to subsequently detect your housekeeper but not a low copy number gene of interest
5. As you say, if you have made sure that the Ta of your gel based PCR or sybr green qPCR is between TM to Tm -5C and invariably Tm-2C and also increased your Mg in 0.5mM increments then you need to consider the placement of your primers; how you carry out your RT reaction and also potential secondary structure in your target:
Take a look at a prior answer of mine
https://www.researchgate.net/post/Should_I_use_random_primer_or_Oligo_dT_ror_relative_quantification_of_gene_expression_in_Q-PCR
5. In essence you need to make sure
- Your primers are within 1KB of the polyA tail if using Oligo dT in your RT reaction
- Better still use Random hexamers in your RT reaction; either with or instead of Oligo DT
- Make sure your amplicon is 100bp-200bp in length (not say 500bp) to maximise efficient qPCR or gel based PCR
- Select a region where predicted target secondary structure does not appear to exist using packages like m fold
http://unafold.rna.albany.edu/?q=mfold/mfold-references
- Similarly, screen your low copy number GOI primers for self or hetero dimers (primer dimers) to ensure efficient amplification with of your low copy number gene using something like IDT oligo tools
https://www.idtdna.com/calc/analyser
See my own word doc I designed for students to maximise (optimise) primer performance (attached)
6. Finally to conclude I would say having made cDNA from at least 1ug of high quality RNA (260/230 > 1.0 and thus devoid of GITC) preferably using random hexamers try amplification of small (~200bp) fragments either using standard PCR @ Tm-2C or as suggested by Giacomo by touchdown PCR, which I have got to work very well (and is especially effective if the Tm of your primers is > 2C)
Ive definitely said enough !!! Good luck !!
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