Are you even a real researcher if you have never cried over a qRT-PCR result? The biggest challenge in setting up a quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) is ensuring that the readings you obtain are accurate.
If you feel like the RT instrument has a grudge against you, do not fret! Much of our focus is devoted to the RNA isolation steps, striving for those perfect RNA bands, or meticulously working on achieving perfect dispensing in the dizzying array of 384-well plates.
Here is a step-by-step guide for the processes BETWEEN RNA isolation and qRT-PCR to ensure that you are proceeding with the best quality RNA that guarantees qRT-PCR success:
1. Isolating the RNA
After isolating RNA using your preferred kit (or the one your lab funds can afford!), it’s crucial to check its quality. You can run the RNA on a formaldehyde agarose gel or obtain the RNA Integrity Number (RIN) (if your lab has crazy expensive resources!).
How to Check RNA Quality Using Electrophoresis
If your RNA is of high quality, you should ideally see three distinct bands: 28S, 18S, and 5S for eukaryotes or 23S, 16S, and 5S for prokaryotes.
However, due to the instability of the 16S RNA in prokaryotes, you may often only see two prominent bands corresponding to the 23S and 5S rRNA.
Tip: When running an agarose gel, you can also detect genomic DNA contamination, which often remains close to the well due to its large size.
How to Check RNA Quality Using the Nanodrop
A nanodrop spectrophotometer can check the absorbance profile of your samples at different wavelengths, which you can use to estimate their quality.
The 260/280 ratio indicates protein contamination, with values around 2.0 being ideal for RNA, while values below 1.8 suggest DNA contamination.
The 260/230 ratio indicates contamination by phenol, salts, or other organic compounds. Ratios close to 2.0 are considered pure, while lower ratios might indicate contamination, especially if using methods like TriZol, which can leave residual contaminants.
How to Check RNA Quality Using the RNA Integrity Number
The RIN is determined using an instrument like the Bioanalyzer, which analyzes your RNA sample in a capillary electrophoresis system. It provides a visual gel image and assigns an integrity score from 1 to 10, with values above 8 indicating high-quality RNA.
2. DNase I Treatment
Eliminating any genomic DNA (gDNA) contamination in our RNA samples is essential, particularly for researchers working with bacterial RNA.
Why is DNase Treatment important?
In eukaryotic RNA, exons (coding regions) are interspersed with introns, which are removed during mRNA processing in a step called splicing.
Primers for qRT-PCR are typically designed to bind across exon-exon junctions, ensuring that only RNA-derived amplicons of 100-200 bp are produced, thus avoiding DNA amplification.
However, bacterial genomes lack introns and exons, meaning primers cannot span exon-exon junctions. As a result, any residual DNA after DNase treatment will be amplified in your qRT-PCR, leading to false-positive results.
Since achieving completely pure RNA devoid of gDNA contamination is nearly impossible with RNA isolation protocols alone, perform DNase I treatment on a specified RNA concentration, typically one µg.
You can scale the reaction according to the amount of RNA required for complementary DNA (cDNA) synthesis.
After this treatment, you must ensure that your RNA hasn’t degraded.
Some DNase I treatment protocols involve heating the RNA mixture at 65°C with EDTA for 10 minutes to deactivate the enzyme.
Since our RNA is minutely (or very) sensitive, this step can be harsh on our RNA. So, we recheck the RNA integrity by running it on the formaldehyde agarose gel.
3. Checking for DNA Contamination
Before synthesizing cDNA, it’s essential to check if most of the contaminating DNA has been eliminated.
Conduct a standard PCR using qRT-PCR housekeeping gene primers on the DNase I-treated RNA. Run a genomic DNA PCR as a control to confirm the absence of gDNA.
Since RNA and DNA have different structures and sequences, the primers are designed to bind specifically to cDNA and, hence, will only bind with remnant gDNA to give amplification.
4. Running a Standard PCR on Formaldehyde Agarose Gel
The PCR reaction mix is run on a 1.5% formaldehyde agarose gel with a marker to check for specific-sized bands indicating the presence of residual DNA.
This step is crucial for ensuring the RNA is free of DNA contamination and contributes significantly to the success of your experiment. A faint band might still appear, which can be expected since it’s nearly impossible to eliminate all DNA contamination.
This is acceptable if the signal is significantly lower than the genomic DNA control, which can be later quantified in your qRT-PCR results. We’ll explain how to do this in the last section.
However, if the bands in your RNA sample are relatively strong compared to your genomic DNA control, you must perform additional DNase I treatment and repeat the PCR-gel analysis process.
Other contaminants, such as leftover phenol and salts, can be detected by 260/230 readings on a Nanodrop spectrophotometer.
A 260/230 ratio close to 2.0 indicates a good-quality sample, while lower values suggest contamination.
You can remove these contaminants by performing a phenol-chloroform extraction followed by ethanol precipitation. A thorough analysis of RNA samples is crucial before proceeding with cDNA synthesis.
5. Preparing RNA for cDNA Synthesis
Your RNA is ready for cDNA synthesis!
Follow the manufacturer’s protocol carefully, ensuring an RNase-free environment and avoiding DNA contamination.
After synthesizing cDNA, perform a standard PCR using your qRT-PCR gene primers to confirm the quality of your cDNA and the specificity of your primers.
If running the mixture on a 1.5% agarose gel produces crisp bands of the expected gene size, it indicates that your cDNA is of good quality and your primers are binding correctly.
Use housekeeping gene primers to check cDNA samples where you expect gene downregulation of your test genes. Choose your primers accordingly based on what you are testing for in each sample.
6. Setting p qRT-PCR
Now, for the moment of truth! When setting up qRT-PCR, your -RT control results should be significantly lower than your +RT results, indicating successful elimination of DNA contamination.
If there is a slight signal in your -RT control, don’t worry—it won’t affect your results as long as it’s several folds lower than the +RT signal.
How It Works
- In the -RT control, the reverse transcriptase enzyme, which converts RNA into cDNA, is omitted during the cDNA synthesis step. Any signal detected in the subsequent PCR should come from residual gDNA rather than RNA.
- If you see amplification in the -RT control, it indicates that DNA contamination is present, and you could compromise the results of your qRT-PCR.
I have a Ct value in my -RT!
If you get a Ct reading in your -RT control, do not fret!
Ct value is the cumulative threshold value, which is the number of cycles required for the fluorescent signal to exceed the background signal.
The lower the Ct value, the fewer the cycles it took to pick up the signal, hence the higher the amount of target gene. You rarely have an undetectable Ct value in your -RT control.
If your +RT value is 23 and -RT is supposed 33, there is a 10 CT difference between the two. So the actual difference in terms of fold is 210, which is 1024-fold lower!
You can take a deep breath because this indicates that the readings are good to go. There is no hard and fast rule as to how much difference in Ct values is considered acceptable.
Any minor contamination will be factored in your qRT-PCR results, mainly using housekeeping genes for normalization. It is important to choose a housekeeping gene that remains stable across the samples. Read this article for more information about the crucial qRT controls you should use
Summarizing the Best Practices for qRT-PCR Setup
While perfecting your technique on 384-well plates is crucial, ensuring high-quality starting material is equally important.
Use a -RT control—where reverse transcriptase is omitted—to check for DNA contamination, but remember, this control doesn’t guarantee success if contamination occurs during RNA isolation.
Implement simple checkpoints between RNA isolation and qRT-PCR to avoid wasting time and resources. With these practices, you’ll ensure high-quality results and only have your pipetting skills to perfect! Stay tuned as we explore that topic in our next article.
Wishing you smooth gradients and clean peaks!
