NZY First-Strand cDNA Synthesis Kit, separate oligos (MB170)

Refrigerated package

196,00857,00

Description: NZY First-Strand cDNA Synthesis Kit, separate oligos, is a system that includes all the necessary components to synthesize first-strand cDNA, except the template RNA. Random hexamers and Oligo(dT)18 primers are provided in separate tubes to offer the convenience to choose the appropriate primer to initiate your reverse-transcription reaction. The resulting single-stranded cDNA is suitable for use in real-time quantitative Reverse Transcription PCR (RT-qPCR). NZY First-Strand cDNA Synthesis Kit, separate oligos, 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. Besides random hexamers and Oligo(dT)18 primers, the kit includes NZYRT 2× Master Mix, no oligos, which contains dNTPs, MgCl2 and an optimized RT buffer; 10× Annealing Buffer and NZYRT Enzyme Mix. The NZYRT Enzyme Mix includes both NZY Reverse Transcriptase (RNase H minus) and 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 reactions since PCR primers will bind more easily to the cDNA

Features:
– Possibility to choose the primer to initiate the reaction
– Provides high yields of full-length cDNA products
– Formulated to increase sensitivity in RT-qPCR
– 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

Specifications:
– Storage conditions: Store all kit components at -20ºC
– Shipping conditions: Shipped with dry ice

Components:
– NZYRT Enzyme Mix
– NZYRT 2x Master Mix, no oligos
– 10× Annealing Buffer
– Random hexamer mix (50 ng/µL)
– Oligo(dT)18 primer mix (50 µM)
– NZY RNase H (E. coli)
– DEPC-treated H2O

Product Brochure EN
Safety Information EN
Informacao de Seguranca PT
Certificate of Analysis ZN082
Certificate of Analysis ZN081
Certificate of Analysis ZN042
Certificate of Analysis ZN041
Certificate of Analysis ZN031
Certificate of Analysis ZN012
Certificate of Analysis ZN011
Certificate of Analysis YO102
Certificate of Analysis YO101
Certificate of Analysis YO041
Certificate of Analysis YL111
Certificate of Analysis YL094
Certificate of Analysis YL093
Certificate of Analysis YL092

1. What primers are used for Reverse Transcription?
There are three different approaches for priming cDNA reactions: oligo(dT) primers, random primers, or sequence-specific primers. These primers differently bind to the template RNA strand, by providing a starting point for the cDNA synthesis, and each one has advantages and disadvantages. The choice between these three priming methods will depend on the size and type of RNA, on the reverse transcription temperature, or on the intended downstream applications. 

RT_priming

An overview of different priming methods for reverse transcription

Oligo(dT) primers specifically bind to the poly(A)-tail found at the 3´-end of most eukaryotic mRNAs. The capacity to generate many different cDNAs from the same starting RNA pool, makes these primers the preferred choice for two-step RT-PCR reactions. Different types of oligo(dT)s are available. Oligo (dT)18 primer mix, a homogenous mixture of 18-mer thymidines, is available at NZYTech for the synthesis of full-length cDNA from poly(A)-tailed mRNA. In contrast to the standard oligo (dT), which randomly bind within the poly(A) tail of the eukaryotic mRNA, the anchored oligo(dT) primers bind at the beginning of the tail. This avoids an unnecessary reverse transcription of this often long region as well as erroneous products synthesized by mispriming.

Random hexamers are preferred for long transcripts or if they contain significant secondary structures. In addition, random hexamers are used for non-polyadenylated target templates (as prokaryotic mRNA). They will perform random priming throughout the entire length of the RNA to generate a cDNA pool containing various lengths of cDNA. With this method, all RNAs present in a population constitute templates for cDNA synthesis experiment. NZYTech provides a Random hexamer mix that includes oligonucleotides representing all possible hexamer sequences.

A mixture of both oligo(dT) and random hexamer primers is usually used to improve the efficiency of cDNA synthesis and qPCR sensitivity.

Gene-specific primers (GSPs) enhance sensitivity by allowing the reverse transcription of a specific RNA sequence. This priming method is chosen to perform one-step RT-PCR reactions once the same primer is used in both the RT and PCR steps. However, GSPs offer less flexibility than oligo(dT) and random primers, since each cDNA synthesis is limited to one target gene.

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.

You may also like…