STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
1. Open the qPCR software.
2. Create a new RT-PCR protocol:
● Select "New", then choose "ABSOLUTE Quantification".
3. Set up the experiment:
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
● In the Setup panel, configure the following:
o Experiment Name:
Example: 20250410_Set 1 B (use the current date followed by the set name).
o Username:
Enter the name of the person or group who performed the PCR.
Example: Group 1
o Comments (optional):
You may enter the target gene or purpose of the experiment, or leave it blank.
Add a detector:
● In the Detector column, input the gene you want to detect.
Example: 16s rRNA
● In the Reporter field, select SYBR.
To perform multiplexing:
● Click "Add Detector" to input additional genes.
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
Sample Column:
1. Click “Batch Add”.
2. Since there are 3 groups, add:
o 3 Unknown samples
o 3 Negative Controls (NC)
o 3 Standards
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
Plate Setup:
● This is where samples
and target genes are organized.
1. Avoid using the
outermost lanes (Row 1 and
Column A)—they’re often
blocked by the heat block and
may not function properly.
2. Start placing samples from Column C (Column 2).
3. Important: Keep Standards well-separated from Negative Controls and Unknown
Samples to avoid cross-contamination.
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
4. To assign a target gene to a sample:
o Use the “Assay Reagent” section and input the gene to detect.
o Example: 16s rRNA
o Note: Do not combine UIDE with SYBR Green—SYBR is non-specific and
only suitable for detecting a single gene.
5. After assigning the gene, go to “Properties” and set the sample type:
o Unknown, Negative Control, or Standard
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
Program Column:
● Adjust the temperature and duration of each stage.
● Add a melting stage to confirm product specificity.
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
o This is essential when using SYBR Green, as it binds to all double-stranded
DNA—including nonspecific products and primer-dimers.
Annealing/Extension Stage – Why is Sampling Checked?
● Sampling enables the machine to measure fluorescence.
● It must be checked during the Annealing/Extension step, where dsDNA forms.
● Do not check sampling during Denaturation, as DNA is single-stranded and
fluorescence cannot be measured.
STEP BY STEP ON USING qPC SOFTWARE.
By: Tuguinay, Tricia Diseree H.
Final Step:
● Once setup is complete, click “Run” to start the qPCR process.