Sanshibio One Step RT-qPCR Kit (Probe)

One Step RT-qPCR Kit (Probe)

Item No：ER101        Specifiation：50T/100T       Storage: Store at -20°C

Product introduction：

This product is a specialized reagent for one-step reverse transcription-real-time fluorescence quantitative PCR (RT-qPCR) using the probe method. Performing One Step RT-qPCR reactions with this product allows for continuous processing within the same reaction tube, simplifying the procedure and avoiding cross-contamination between samples while enhancing detection sensitivity. This assay kit employs a novel reverse transcriptase (HI-MMLV Reverse Transcriptase), a newly designed antibody-modified hot-start Taq DNA polymerase, and RNase Inhibitor in the form of the One Step RT-qPCR Enzyme Mix. It features enhanced RNA affinity, thermal stability, higher amplification efficiency, and specificity. This enables the generation of robust standard curves within a wide quantitative range, facilitating accurate quantification of various target genes at different expression levels. It offers good repeatability and high reliability.

Product contents：

Storage:

Store at -20°C, with a minimum shelf life of 12 months.

Activity Definition:

Using activated mahi-mahi sperm DNA as template/primer, the activity is defined as 1 unit (U) of acid-insoluble material incorporated, by taking up 10 nmol of nucleotides within 30 minutes at 74°C.

Product Uses:

This assay kit is suitable for probe-based one-step reverse transcription-real-time fluorescence quantitative PCR. It allows accurate and straightforward analysis of RNA expression, particularly well-suited for detecting small amounts of RNA. It is compatible with various types of fluorescence quantitative PCR instruments.

Note：For certain companies’ Real-Time PCR amplification instruments that exhibit inter-well fluorescence signal discrepancies, please either prepare or contact the manufacturer to acquire ROX Reference Dye to correct these errors. Detailed specifics should be chosen based on experimental design, instrument manuals, or the specific usage requirements of various fluorescent probes.

Additionally, this assay kit does not include a gDNA (genomic DNA) removal component. If your experiment requires freedom from gDNA interference, please conduct a gDNA removal step during or after RNA extraction and then proceed with the experiment using this assay kit. This precaution will prevent any potential interference with your experimental results.

Operating steps:

1. Prepare the following reaction mixture in RNase-Free centrifuge tubes:

Note：The amounts of each component can be adjusted according to actual needs.

• Perform the One Step RT-qPCR reaction under the following conditions:

• After the reaction is completed, verify the amplification curve of the Real Time PCR instrument and analyze the results.

Precautions:

1. The One Step RT-qPCR Enzyme Mix contains a high concentration of glycerol. Before use, please briefly centrifuge the mix to collect it at the bottom of the reaction tube. Gently pipette up and down to mix thoroughly. For reaction setup, use RNase-Free pipette tips, EP tubes, etc., and avoid contamination as much as possible.
2. Typically, a primer final concentration of 0.2 µM is sufficient for optimal amplification. When the reaction performance is suboptimal, adjust the primer concentration within the range of 0.1 to 1.0 µM. Probe concentration can be adjusted between 50 nM and 250 nM. qPCR is highly sensitive, and the accuracy of template amount during reaction setup greatly influences the final quantification results. It is recommended to dilute the template (e.g., dilute 2-5 µL of sample) before adding it to the reaction mix to enhance experimental reproducibility.
3. Choose an amplification product length within the range of 80 bp to 200 bp. For templates with complex secondary structures and high GC regions, raising the reverse transcription temperature to 55°C can improve amplification efficiency and detection sensitivity.
4. Verify whether the actual Real Time PCR instrument used supports fast amplification cycles. For initial attempts, conduct preliminary experiments to confirm. Adjust the extension time according to the shortest data collection time limit required by the specific Real Time PCR instrument used. For instruments like ABI 7700 and ABI 7900, use at least 30 seconds. For ABI 7000 and ABI 7300, use at least 31 seconds. For ABI 7500, use at least 34 seconds. If using the ABI series of Real Time PCR instruments, include ROX Reference dye in the solution preparation.