Description: NZY First-Strand cDNA Synthesis Kit is a system that includes all the necessary components to synthesize first-strand cDNA, except the template RNA.The resulting single-stranded cDNA is suitable for use in real-time quantitative Reverse Transcription PCR (RT-qPCR). NZY First-Strand cDNA Synthesis Kit is formulated to provide high yields of full-length cDNA products and to increase sensitivity in RT-qPCR. Starting material can range from 10 pg up to 5 µg of total RNA. The kit includes a combination of random hexamers and oligo(dT)18 primers in order to increase sensitivity. The primers are included in the NZYRT 2× Master Mix, which also contains dNTPs, MgCl2 and an optimized RT buffer. NZYRT Enzyme Mix includes both the NZY Reverse Transcriptase (RNase H minus) and the NZY Ribonuclease Inhibitor in order to protect RNA against degradation due to ribonuclease contamination. RNase H (from E. coli) is provided in a separate tube to specifically degrade the RNA template in cDNA:RNA hybrids after the first-strand cDNA synthesis. This procedure will improve the sensitivity of subsequent RT-qPCR reaction since PCR primers will bind more easily to the cDNA.
Features:
– Provides high yields of full-length cDNA products
– Formulated to increase sensitivity in RT-qPCR
– Primer type: oligo(dT)18 and random hexamers
– Starting material: 10 pg to 5 µg of total RNA
– Thermostable – working temperature range 37-50 ºC
– Convenient and reliable
Applications:
– First-strand cDNA synthesis for use in real-time quantitative RT-PCR (RT-qPCR)
– Two-step RT-PCR assays
– Ideal for high GC-content templates due to the optimum activity at 50 ºC
High quality cDNA with low levels of starting RNA:
NZY First-Strand cDNA Synthesis Kit is an ideal choice for reverse transcription of low abundant RNA templates. NZYRT Enzyme Mix present in the kit shows high sensitivity, allowing synthesis of a wide range of template amounts.
A10-fold serial dilution of total RNA from mouse liver (1 μg to 10 pg) was reverse transcribed in a 20 μL reaction using the NZY First-Strand Synthesis Kit.1 μL of the resultant cDNA was then used as template in an end-point PCR using specific primers for amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene with Supreme NZYTaq II DNA polymerase. NC: No template control. Lane M: NZYDNA Ladder III.
Specifications:
– Storage conditions: Store all kit components at -20ºC
– Shipping conditions: Shipped with dry ice
Components:
– NZYRT Enzyme Mix
– NZYRT 2x Master Mix
– NZY RNase H (E. coli)
– DEPC-treated H2O
Reverse Transcription kits available at NZYTech: Selection Guide
2. Should I treat the synthesized cDNA with RNase H before PCR?
Addition of RNase H after first-strand synthesis will degrade the RNA used as template, by removing it from the cDNA:RNA hybrid molecule. The RNA is still present when using RNase H– versions of reverse transcriptase, as is the case of the NZYReverse Transcriptase. Presence of RNA during PCR could inhibit annealing of the primers to the cDNA and then affect the amplification reaction, especially for long fragments. However, the 95°C denaturing step could cause RNA degradation of the RNA-cDNA hybrids and therefore RNase H treatment may not be necessary. We recommend performing RNase H digestion before PCR when using lower levels of template or when amplifying long fragments.
3. How much synthesized cDNA should be used in a PCR reaction?
Do not exceed 10% of the final PCR reaction volume. The volume of cDNA used will depend on the amount of RNA used as template for first-strand synthesis, as well as the abundance of the target gene.
4. Little or no RT-PCR/RT-qPCR amplification product is observed. What should I do?
This may result from several factors, such as:
a) RNA damage or degradation. Analyze RNA by denaturing gel electrophoresis to verify nucleic acid integrity. Use aseptic conditions while working with RNA to prevent RNase contamination. Replace RNA if necessary.
b) Presence of RT inhibitors. Some inhibitors of RT enzymes include: SDS, EDTA, glycerol, sodium phosphate, spermidine, formamide and guanidine salts. They can be problematic if present in smaller reaction volumes. If necessary, remove inhibitors by ethanol precipitation of the RNA preparation before use; wash the pellet with 70% (v/v) ethanol.
c) Not enough starting RNA. Increase the concentration of starting RNA.
5.What to do when unexpected bands are observed after electrophoretic analysis of PCR products using the synthesized cDNA as template?
This may result from several factors, such as:
a) Contamination by genomic DNA. To test if products were derived from DNA, perform a no-RT control during first-strand cDNA synthesis. If amplification products are detected after the PCR reaction in the absence of reverse transcriptase, it may be necessary to eliminate residual genomic DNA from the RNA sample. A RNAse-free DNase may be used (pre-treatment RNA). The DNase volume should not exceed 10% of the total reaction volume.
b) Non-specific annealing of primers. Adjust annealing conditions and/or use a DNA polymerase with hot-start capacity (e.g. Supreme NZYTaq II DNA polymerase, cat. No. MB355). Optimize primers and magnesium concentration.
c) Primer-dimers. Adjust annealing conditions and/or design another set of primers without complementary sequences. Include a positive control in the PCR, i.e. a template that will be correctly amplified with the primers that you designed.
