Gisai Bio providesRT-qPCR kits; the products include reverse transcription and quantitative PCR compatible reagents and can be used for detection and analysis of circRNA expression levels and differences, and are also suitable for mRNA, lncRNA, etc. In addition,Gisai Bio can also provide separate reverse transcription kits andquantitative PCR detection reagents for flexible use by customers.
This product’sTransScript RT Enzyme (M-MLV) is a novel reverse transcriptase obtained from the M-MLV (RNase H-) Reverse Transcriptase backbone by in vitro molecular evolution techniques. It has greatly enhanced thermal stability, with a half-life at 50°C exceeding 240 minutes, and remains stable during prolonged reactions at 55°C, making it well suited for reverse transcription of RNA templates with complex secondary structures. The reagents for quantitative PCR detection are specialized reagents for qPCR using SYBR Green I intercalating fluorescence.circRNA and lncRNAhave relatively low expression abundance,requiring optimization of multiple conditions to refine the amplification system.This productcombined with a buffer optimally formulated for qPCR,can effectively suppress nonspecific amplification and significantly improve amplification efficiency, making it suitable for high-sensitivity qPCR reactions.
Product Features
1. The kit contains Oligo dT and random primers, providing broad applicability;
2. The system is optimized for low-copy RNA, offering high amplification efficiency, strong specificity, and high sensitivity;
3. Comprehensive supporting primer products and technical services: RIBOBIO, leveraging years of technical experience, has validated over 10,000 pairs of circRNA primers, screened more than 2,000 specific primer pairs, and established a “circRNA Primer Library.” We can provide researchers with primer sequence information and offer circRNA qPCR primer design and circRNA qPCR testing services to effectively ensure rapid experimental success.
Customer Article
[1] IF13.493 The circFASN/miR-33a pathway participates in tacrolimus-induced dysregulation of hepatic triglyceride homeostasis. Signal Transduction and Targeted Therapy
[2] IF13.493 The circFASN/miR-33a pathway participates in tacrolimus-induced dysregulation of hepatic triglyceride homeostasis. Signal Transduction and Targeted Therapy
[3] IF9.229
In Vitro Study on the Piezodynamic Therapy with a BaTiO3-Coated Titanium Scaffold under Low-Intensity Pulsed Ultrasound Stimulation. ACS Applied Materials & Interfaces
[4] IF9.229
In Vitro Study on the Piezodynamic Therapy with a BaTiO3-Coated Titanium Scaffold under Low-Intensity Pulsed Ultrasound Stimulation. ACS Applied Materials & Interfaces
[5] IF8.886 Effects of long-term culture on the biological characteristics and RNA profiles of human bone-marrow-derived mesenchymal stem cells. Molecular Therapy-Nucleic Acids
Q & A
A Frequently Asked Questions
1. How do you set the temperature for the melt curve?
When we perform the test we useABI
a 7500 instrument; the melt curve settings are the instrument defaults, so use the instrument defaults for the melt curve.
2. Can kit GS0202-2 be used only for reverse transcription of circRNA?
No, it can also be used for conventionalmRNA and lncRNA reverse transcription.
3. Does the Reverse Transcription Primer include both Oligo dT and random primers, or does it contain only one type of primer?
IncludesOligo dT and random primers.
4. What are the advantages of our fluorescent quantitative PCR kits?
High amplification efficiency and good specificity.
5. When preparing qPCR reaction mix, with a reaction volume of 20 µl, how much template should be added?
It is recommended to use
1 µg of RNA for reverse transcription; dilute the resulting cDNA 5–10 fold and use 2 µl of cDNA for the qPCR reaction. Appropriate adjustments can be made according to the customer’s experimental requirements.
6. Can the GS0202-2 kit be used for reverse transcription of cfRNA?
Okay.
7. What information do we need to provide for the primer design and synthesis for the GS0202 kit?
You need to provide the species information, the relevantcircRNA ID numbers and sequences.
8. Can the master mix inside be run directly on electrophoresis, or is loading buffer still needed?
Loading buffer is required.
9.CT value appears too late or the test is negative
a. Very low amplification efficiency: optimize reaction conditions and try a three-step amplification program.
b. Template concentration too low: reduce dilution and repeat experiments; for samples with unknown concentration, generally start from the highest concentration.
c. Template degradation: reprepare the template and repeat the experiment.
10、No amplification curve appears at the end of the reaction
a. circRNA is not expressed in the cell line or tissue: change the template.
b. Insufficient number of reaction cycles: the cycle number is generally set to 40, but note that too many cycles will increase background signal and reduce the reliability of the values.
c. Confirm whether a signal acquisition step is set in the program: for two-step amplification programs, signal acquisition is generally set at the annealing/extension stage; for three-step amplification programs, signal acquisition should be set at the 72°C extension stage.
d. Template concentration too low: reduce the dilution and repeat the test; for samples with unknown concentration, generally start from the highest concentration.
e. Template degradation: reprepare the template and repeat the test.
11. Significant amplification also appears in the negative control
Reaction mix or water contaminated: replace with new one
Repeat the Mix or water control. Prepare the reaction system inside a laminar flow hood to reduce aerosol contamination.
12. Poor experimental reproducibility
a. Inaccurate pipetting volumes: Use high-performance pipettes, increase the reaction volume, and dilute the template at higher folds so that a larger volume is added to the reaction system.
b. Inconsistent temperature control at different positions of the qPCR instrument: Calibrate the instrument regularly.
c. Template concentration too low: the lower the template concentration, the poorer the reproducibility; use a less diluted template or increase the sample volume.
13. Can this product be stored at 4°C
a. No. Storage at 4°C will lead to decreased product activity.
b. This product can maintain activity long-term when stored at -20°C; storage at -20°C is recommended.
c. Repeated freeze–thaw cycles may also lead to decreased product activity; therefore, when each usage amount is small, it is recommended to aliquot into small volumes and store at –20°C.
B Precautions
1. Avoid repeated freeze–thaw cycles of this product as far as possible to prevent loss of enzyme activity. If the amount used each time is small, it is recommended to aliquot into small portions for use.
2.
Before use, invert several times to mix; do not vortex to avoid generating excessive bubbles that may cause inaccuracies in the reaction system and thus affect quantification results. After mixing, briefly centrifuge and then use. If bubbles are inadvertently generated during handling, centrifuge again before use.
3. Because the 2×qPCR SYBR Green Master Mix in the product contains the fluorescent dye SYBR Green I, exposure to strong light should be minimized both when storing the Mix and when preparing the reaction system.
