Quantitative Real-Time PCR (qRT-PCR)
Experimental Gene(s) Selection
In order to perform qRT-PCR, one needs to both identify experimental and housekeeping genes and design primers for them. Start by looking up your plant under the stress condition you want to test. For example, I want to do a drought stress experiment with potatoes so I internet searched for drought tolerant genes in potato and found a paper that used STANN1.
You will want to find multiple genes for the given scenario. You can not rule out the possibility of working with an unknown gene or a lesser known gene. Maybe a lot of work has not been done with your plant or you cannot find the gene sequence you want to evaluate. Start by looking at closely related relatives and see if you can find the gene sequence there.
Research housekeeping genes to use
Next, you will need to find endogenous genes, also called reference, control genes or housekeeping genes, that will not be affected by your experiment. Housekeeping genes will not work with all experiments! Sometimes your housekeeping gene is upregulated by your experiment or by the age of your plant material.
For example, I internet searched housekeeping genes potato and found these housekeeping genes were used.
- Ubiquitin gene is useful in some hosts
- Elongation factor 1-alpha (ef1alpha)
- 18S rRNA
- Adenine phosphoribosyl transferase (aprt)
- Cytoplasmic ribosomal protein L2
Most common genes used are actin, beta tubulin, and ubiquitin. Many papers use glyceraldehyde 3 phosphatase dehydrogenase (GADPH) but it all depends on the host you are working with.
You should run your own test of the endogenous genes you are planning to use to first determine whether or not your housekeeping/control gene will serve the purpose you are looking for (that it doesnâ€™t vary between samples or treatments). When you test the amplification of this gene, all your samples from different treatments should have results that are roughly the same.
A good review paper on housekeeping genes
Look at some of the pitfalls of qRT-PCR and may provide some insight on how to design your experiment (or keep you up late with worry).
The Applied Biosystems Manual on StepOnePlus Systems.
Applied Biosystems Guide to Relative Quantification using real-time PCR.
Primer design and verification
For many plants or microbes, someone else has already discovered what primers will amplify specific genes. They may have already performed qRT-PCR with the primers and determined which primers work best. Take advantage of that but do not trust it blindly. Even if someone else has discovered the primers, you still must validate the sequence.After you find the genes you want to use, you need to look up their sequence on NCBI, save the sequence to a .txt document on a thumb drive in FASTA format, then run it through primer design software (Primer Express software is available at the Core Facility – free to use).You can use the Primer Express software in the core facility room with help from Mike.
Once you have settled on your primer sequences, order your primers through Rutgers market place under Thermofisher scientific, direct ordering, custom primers (oligonucleotides), DNA Value Oligos, Basic and Bulk upload. They are around $5 each and arrive after a few days. A more economical and quicker way to get your primers is through Integrated DNA Technologies at RWJMS (Busch Campus, Piscataway). They sell primers around $2.70-3.00 and you can pick up your order around 24 hours after ordering the primers in person at the RWJ Medical School Building or they deliver to you. Saves on shipping and avoiding on waiting for shipments.
Now is a good time to extract some genomic DNA (gDNA) from your plant while you wait for your primers to arrive. Test your primers to make sure they are really going to work for you by running a basic PCR.
After extracting DNA dilute to about 5 ng/ul before running PCR. You can also dilute 10 ul of DNA into 990 ul for 100 fold dilution then serial dilute to -6 then run PCR with each dilution and see if the primers amp your gene. Even though there are introns in the DNA (meaning the size might vary), this validation step works in most situations. Every once in a while you may have to do this with the converted DNA (cDNA) if you fail to amplify with repeated attempts using gDNA.
- DNeasy Powerplant Pro kit by Qiagen catalog number: 13400-50
- Items needed that are not provided in the kit:
- Liquid Nitrogen
- Sterile mortar and pestle
- PCR Super Mix from supply center catalog number: 10572014
Remember RNA is not stable like DNA. You need to be extra careful. Sterilize tools, work in a hood, wear a lab coat and always wear gloves (if you touch your skin or clothing with your gloves – change them to avoid contaminating your extract with RNase).
Note: The tissue required for RNA extraction is 0.03 g or less.
- RNeasy mini kit from Qiagen catalog number: 74104 (50) or 74106 (250)
- UltraPure DNAse/RNAse-free distilled water from the freezer program catalog number: 10977015
Additional items needed:
- 10 ul beta-mercaptoethanol, or 20 ul 2 M dithiothreitol (DTT)
- Several Eppendorf tubes
- Liquid Nitrogen
- Sterile mortar and pestle
You may want to consider purchasing a DNA removal kit which may remove any DNA from your RNA extraction. The main concept is to use a DNAse. For example, you could use the following kit or similar kits to remove DNA.
Assessing RNA quality
Assess the quality of your RNA using the NanoDrop Spectrophotometer (2nd floor facility). You need keycard access. Make sure you change the settings of the NanoDrop software to measure RNA instead of DNA. First load a blank. Then load 1-2 ul samples into the NanoDrop. For RNA you are looking for a ratio of ~2.00 for 260/280. You must start with good quality RNA otherwise you may compromise the results of the qRT-PCR.
You may want to consider starting with RNA samples from different treatments that have the same concentration of RNA prior to doing the reverse transcription.
Reference on RNA quality assessment
Decide whether you want to do a 1-step or a 2-step qRT-PCR. The Applied Biosystems manual has a good explanation of the difference between both.
Convert RNA to cDNA
High capacity cDNA reverse transcription kit from Thermofisher Scientific catalog number: 4368814
What is the time range for converting RNA to cDNA?
If you can not convert RNA to cDNA right away, you can store overnight in -20 C. It is risky to delay this step so plan ahead if you can to convert your RNA the same day (start early). Once you convert RNA to cDNA, the cDNA can be stored for longer periods of time compared to RNA samples.
How to set your PCR machine to run 1 cycle
The minimum number of cycles our PCR machine can do is two so this is how you need to set up your machine to complete the one cycle into two cycles.
- 25 C for 1 min
- 25 C for 10 mins
- 37 C for 60 mins
- 85 C for 5 mins
85 C for 5 mins, then Cool 4 C
Talk with Mike about your experiment, what genes you are using for reference and experiment. He will design the layout of your well plate (or collection of strip tubes) and print you out a map so you know what to pipet into each well. If you have a windows PC you can download StepOne Software and prepare the layout of your plate/strip tube prior to actually doing the assay. If you do this ahead of time you can save the layout on a USB and take it to the Core Facility where it can be easily loaded onto the software there. You should also have an idea when you want to setup and run the qRT-PCR so you can reserve the time on the machine. You want to run each sample in triplicate.
Some things you will be asked by Mike.
- How many genes are you looking at?
- What housekeeping genes are you using?
If you have room on the plate it may be in your best interest do try different dilutions of your template cDNA. Mike may suggest you dilute a small amount of cDNA 50x = 5 ul of cDNA template into 245 ul of DNAse/RNAse-free distilled water.
Setting up standard curve
Whatever dilution you are using in the rest of the experiment will be your starting point for this part too. So if you are diluting 50x you would make sure you dilute enough cDNA for your curve. Read about more about setting up a standard curve.
Supplies for qRT-PCR
Power SYBR green from (supply center catalog number: 4367659)
Well plate vs. strip tubes
- Well plates (supply center several options)
- Requires plate sealer and sealer tool
- Less chance for contamination while sealing the tubes
- Plate is well labeled
- Strip tubes (supply center: 4358293)
- Less expensive
- Requires a tube holder
- Requires optical strip caps (supply center: 4323032)
- Easy to replace a strip if you make an error loading your sample
- Must label strip caps 1-12 to keep tubes in order (tab at end of cap strip) Do not write the names of the samples on the tubes or on the caps.
- Increased chance of contamination
In either case, it is good to use some color to help remind you where specific treatments go as we did when working with strip tubes.
qRT-PCR master mix protocol 1x
- 10 ul Power SYBR
- 1 ul Forward primer (200 to 250 nM final concentration)
- 1 ul Reverse primer (200 to 250 nM final concentration)
- 3 ul DNase and RNase free water
Add 15 ul of master mix and 5 ul of template to each tube for a total of 20 ul per tube. For the No Template Control (NTC) add 15 ul master mix and 5 ul of water. Remember that you want to run each sample in triplicate.
The Power SYBR will be frozen when it comes out of the freezer, you need to make sure it completely melted and invert several times to mix before you make your master mix.
Give yourself ample time so you can pipet consistently, start at least 2.5 hours before your scheduled time on the machine. Only use really good pipettes. If possible arrive 5 mins early because your plate/tubes will need to be spun by Mike.
Mike will help you through the setup process. Do not be afraid to ask him questions. Schedule time to speak with him in case he is busy.
Why melt curves are important
The melt curve is run by the qPCR machine after all of the cycles have been completed. The importance of the melt curve analysis is to determine that only a single PCR product has been generated. A melt curve analysis can increase the run-time of your entire experiment. You can change the melt curve portion of the run to 1.0C instead of 0.3C which may be the default that the machine runs it at. 1 clean peak typically indicates the presence of a single amplicon which is what you are looking for.