How to Check the Success of Your cDNA Synthesis: Simple Validation Methods for Reliable Gene Expression Studies

 How to Confirm Successful cDNA Synthesis in Plasmodium falciparum: Best Housekeeping Genes to Use

If you've just synthesized cDNA from Plasmodium falciparum RNA, the next essential step is confirming that your reverse transcription worked. One of the most reliable ways to do this is by amplifying a housekeeping gene—a gene that's stably and consistently expressed across parasite stages.

In this post, we’ll look at the best housekeeping genes commonly used for P. falciparum cDNA quality control, what makes them reliable, and how to choose the right one for your experiment.

What Are Housekeeping Genes and Why Use Them?

Housekeeping genes encode proteins essential for the basic functioning of the cell and are usually expressed at constant levels in most biological conditions. In Plasmodium falciparum research, they are routinely used as internal controls to:

• Confirm cDNA synthesis success

• Normalize gene expression in qPCR

• Detect RNA degradation or contamination

Top Housekeeping Genes for P. falciparum cDNA Validation

Here are commonly used genes you can amplify to check your cDNA:

Gene Name	Function	Why It’s Useful	PlasmoDB ID
PfSeryl-tRNA synthetase	Adds serine to tRNA during translation	Stable expression across stages	PF3D7_0717700
PfAldolase	Glycolysis enzyme	High expression, well-conserved	PF3D7_1444800
PfActin I	Cytoskeletal structure	Widely used across species	PF3D7_1246200
GAPDH	Glycolysis enzyme	Common reference gene in eukaryotes	PF3D7_1462800
HSP70	Heat shock protein	Medium-high, stable expression	PF3D7_0818900
18S rRNA	Ribosomal RNA	Very abundant, but no poly-A tail	Multiple

A Few Notes Before You Start PCR:

• Priming matters: If your cDNA synthesis used oligo-dT primers, then 18S rRNA may not amplify well because it lacks a poly-A tail.

• Check for splicing: Choose genes with few or no introns, or design primers that span exon-exon junctions to avoid amplifying residual genomic DNA.

• PfSeryl-tRNA synthetase is a top choice for normalization in P. falciparum studies due to its stable and consistent expression.

Additional cDNA Quality Control Steps

Control Type	Principle/Purpose	Procedure	Interpretation
Checking for Genomic DNA (gDNA) Contamination
No-RT Control (Minus Reverse Transcriptase)	To ensure the PCR signal originates from cDNA (derived from RNA) and not from contaminating genomic DNA (gDNA). Crucial for accurate gene expression studies.	1. Perform a reverse transcription (RT) reaction setup as usual for your samples, but omit the reverse transcriptase enzyme. 2. Use this "no-RT" sample as the template in a subsequent PCR reaction with primers for your gene of interest (or a housekeeping gene). 3. Primer Design (Recommended): Use primers that span an exon-exon junction. This means if gDNA (with introns) is present, the amplicon will be significantly larger or fail to amplify under standard conditions for shorter cDNA amplicons. Alternatively, design one primer in an exon and the other in an adjacent intron (this will only amplify gDNA).	No amplification: Great! This indicates no significant gDNA contamination. Any signal in your actual cDNA samples (those with reverse transcriptase) is genuinely from RNA. Amplification:  Uh-oh. This suggests gDNA contamination in your original RNA sample, or that any DNase treatment was insufficient. Results from cDNA samples showing this contamination in the no-RT control are unreliable for RNA quantification.
General PCR Controls
No Template Control (NTC)	To check for contamination in PCR reagents (e.g., primers, polymerase, dNTPs, buffer) or to detect primer-dimer formation.	Prepare a PCR reaction containing all necessary reagents (primers, polymerase, dNTPs, buffer) but substitute sterile, nuclease-free water for the cDNA template.	No amplification: Perfect. Your reagents are clean, and there are no significant primer-dimers. Amplification: Indicates contamination of one or more PCR components or significant primer-dimer formation. Troubleshoot by testing individual reagents or redesigning primers if necessary.
Positive Control	To confirm that the PCR reagents, equipment, and reaction conditions (including primers and polymerase activity) are working correctly.	Use a sample known to contain the target cDNA sequence as the template. This could be: - cDNA from a cell line or tissue known to express the gene at detectable levels.  - A plasmid containing the cloned target sequence.  - Previously validated cDNA.	Clear amplification of the expected size (e.g., a visible band on a gel or an appropriate Ct/Cq value in qPCR): Excellent. This confirms your PCR setup is functioning as expected. No or weak amplification: Suggests a problem with the PCR setup itself (e.g., degraded enzyme, incorrect primer concentration, faulty thermocycler, or issues with the positive control template).

Final Tip

Always run your PCR product on a gel to confirm expected size and quality. If you’re planning downstream qPCR or expression analysis, validating your housekeeping gene choice upfront can save you significant troubleshooting later.

May 13, 2025 Tags : cDNA quality check , cDNA synthesis , gene expression analysis , housekeeping genes , lab protocols , molecular biology techniques , PCR for cDNA , qPCR validation , reverse transcription , RNA to cDNA , validate cDNA

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