A qPCR Method to Distinguish between Expression of Transgenic and Endogenous Copies of Genes

Materials and reagents

Biological materials

1. Gene synthesis; GeneArt Gene Synthesis (Thermo Fisher Scientific, UK)

Laboratory supplies

1. RNeasy Plant Mini kit (Qiagen, catalog number: 74904)

2. RNase-free DNase set (Qiagen, catalog number: 79254)

3. Bpi1 (Bbs1) restriction enzyme (Thermo Fisher Scientific, catalog number: ER1011)

4. qPCRBIO cDNA synthesis kit (PCR Biosystems Ltd, catalog number: PB30.11-10)

5. SuperScript^TM IV First-Strand Synthesis System (Thermo Fisher Scientific, catalog number: 18091050)

6. qPCR SyGreen Mix (PCR Biosystems Ltd, catalog number: PB20.12-05)

7. qPCR plates (Applied Biosystems, Thermo Fisher Scientific, catalog number: 4346906)

8. Microamp Fast Optical 96-well reaction plates with barcode (Thermo Fisher Scientific, catalog number 4346906)

9. Adhesive PCR plate seals (Thermo Scientific, catalog number: AB-0558)

Equipment

1. MiniAmp thermal cycler (Thermo Fisher Scientific)

2. StepOnePlus real-time PCR system (Applied Biosystems, Thermo Fisher Scientific)

3. NanoDrop (Thermo Scientific, Labtech, ND1000, Thermo Fisher Scientific)

Software

1. Construct design and sequence analysis was carried out using SnapGene software (www.snapgene.com); a plasmid editor, ApE, is a suitable freely available alternative (Davis and Jorgensen, 2022)

2. qPCR data analysis was performed using Thermo Fisher Data Connect Design & Analysis Software

Procedure

1. Transgene design

   Key to this method is the removal of a Bpi1 restriction site from the coding region of the gene that is to be expressed as a transgene. This is frequently required for Golden Gate cloning (Engler et al., 2008; Weber et al., 2011) and has been described in previous protocols (Engler and Marillonnet, 2014). For other cloning methods, any suitable restriction site(s) can be removed.




2. RNA extraction, cDNA synthesis, qPCR

   1. This step is only relevant if using the inducible system as in Bezodis et al. (2022). This protocol may be used for any system in which a transgenic copy of an endogenous gene is expressed.

      Our validation experiments on Arabidopsis thaliana (described below) utilised an inducible expression system active in inflorescences (Bezodis et al., 2022; Schubert et al., 2022). At least four biological replicates are recommended for achieving highly reproducible results.

      1. Select Arabidopsis plants with at least four inflorescence apices; mark two inflorescences for pre-induction and two for post-induction expression, with small pieces of tape of different colours.

      2. Collect the two pre-induction inflorescence apices, harvesting all buds that have not clearly been fertilised. Snap freeze in liquid nitrogen and store at -80 °C until all samples are ready for RNA extraction.

      3. Apply induction as described (Schubert et al., 2022) to the other two marked inflorescences, harvested 18 h after induction.

   2. Extract RNA using the method appropriate for the system being used. For the work in which this method was developed, RNeasy Plant Mini kit with on-column DNA digest was used, following manufacturer’s recommended methods (Note 1).

   3. Synthesize cDNA from the resultant RNA. The qPCRBIO cDNA synthesis kit and SuperScript IV Reverse Transcriptase have been tested; the resultant cDNA, following manufacturer’s recommended protocol, proved suitable for use with this method.

   4. cDNA digestion:

      1. Determine approximate cDNA concentration by NanoDrop (Note 2).

      2. Dilute cDNA to 250 ng/μL in molecular biology grade water (Note 3).

      3. Digest cDNA in 96-well PCR plates in 20 μL reactions as below (Table 1):

         
         

         Table 1. Components for cDNA Restriction Digest

         Component	Volume
         250 ng/μL diluted cDNA	14 μL
         Bpi1	4 μL
         10× digestion buffer	2 μL

         
         

         Mix by pipetting and spin down plates in a plate centrifuge at ~1,200× g for 30 s.

      4. Perform digestion in a thermocycler as below, with lid temperature 103 °C (Table 2).

         
         

         Table 2. Thermocycler run for cDNA Restriction Digest

         Thermocycler step	Step 1	Step 2	Step 3
         Duration	8 h	20 min	hold
         Temperature	37 °C	65 °C	4 °C




3. Primer design and qPCR

   1. Primer design

      1. Primers are needed to amplify an endogenous control housekeeping gene and the gene of interest. The amplification product should be in the region of 70–200 bp with an annealing temperature of > 60 °C.

      2. For the housekeeping gene, such as ubiquitin or tubulin, the region amplified should not contain a Bpi1 site (or other restriction enzymes being used), whilst for the gene of interest the region amplified must contain a region that contains a Bpi1 (or other restriction enzyme) site in the endogenous copy that has been removed as described in section A. Selection of housekeeping genes as controls for qPCR has been discussed previously (Czechowski et al., 2005) and depends on the aims of the specific experiment and tissue being used (Dekkers et al., 2012). Protocol validation by Bezodis et al. (2022) used AtUBQ10 (AT4G05320), which is more stable than some other commonly used reference genes (Czechowski et al., 2005).

      3. Once a suitable region for amplification has been chosen, optimal primers can be designed using primer3 (primer3.ut.ee) (Untergasser et al., 2012).

   2. qPCR reaction

      1. Perform qPCR in Microamp Fast Optical 96-well reaction plates with Adhesive PCR plate seals in either 10 or 20 μL reactions; for the latter, volumes below should be doubled (Table 3).

         
         

         Table 3. Components for qPCR

         Component	Volume
         Digested cDNA	1 μL
         2× SyGreen qPCR Master Mix	5 μL
         10 μM forward primer and 10 μM reverse primer stock solution	1 μL
         Molecular biology grade water	3 μL

      
      

      2. Perform qPCR on a StepOnePlus real-time PCR system programmed as shown below (Table 4). A melt curve is used to check primer specificity. Lid temperature set to 103 °C.

         
         

         Table 4. Real-time PCR system run for qPCR

         Thermocycler step	Step 1	Step 2
         Duration	3 min	15 s	30 s
         Temperature	95 °C	95 °C	60 °C
         Number of cycles	1	40

Data analysis

qPCR data are analysed either by manual calculation using Excel or more easily with Thermo Fisher Data Connect Design & Analysis Software, which allows quantification of expression using the 2^-ΔΔC(T) method (Livak and Schmittgen, 2001). The level of expression of the transgene from digested cDNA is normalised to the level of housekeeping gene used. As described, it is important that the region of the housekeeping gene amplified does not include a restriction enzyme target and, if properly designed as described, the analysis will be no different to any other qPCR experiment. It is important to use the number of biological replicates as the sample size for statistical analyses, as the technical replicates should be averaged and used only to ensure values from the same sample are similar. Various methods of analysis of qPCR data are available, which have been reviewed by Pabinger et al. (2014). Application of this protocol does not change the data analysis.

Notes

1. Numerous kits from different manufacturers are available and method should follow manufacturer’s recommended protocol.

2. Sufficient cDNA is needed for at least six qPCR reactions to allow the amplification of a control housekeeping gene for normalisation and the transgenically expressed endogenous gene of interest, at least in triplicate for technical replicates. This is in addition to the biological replicates described in point B1.

3. This concentration must be lower than the concentration of the most diluted sample; otherwise, RNA extraction and cDNA synthesis should be repeated.

Validation of protocol

Data validating this protocol can be found in our preprint Bezodis et al. (2022) deposited on bioRxiv at doi.org/10.1101/2022.09.15.508103; relevant data are shown in Figure 1D–1G.

Acknowledgments

We thank Vinay Shukla for help with operation of the qPCR machine and data analysis methods and Jasmine Staples for help designing the graphical overview. This work was carried out and funded as part of an Mbiol masters project in the Department of Plant Sciences (now Department of Biology), University of Oxford.

Competing interests

We have no competing interests.

References

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