NZY Reverse Transcriptase (MB124)

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Description: NZY Reverse Transcriptase is a modified recombinant form of the Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase purified from Escherichia coli. The enzyme has been modified in order to promote stability. NZY Reverse Transcriptase synthesizes the complementary DNA strand in the presence of a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template in a wide range of temperatures (37-50°C). This is useful when using templates with high GC-content or with high degree of secondary structure. The enzyme lacks 3´→5´ exonuclease activity and has no RNase H activity, enabling improved synthesis of full-length cDNA even for long mRNA, using random priming.

Features:
– Synthesizes of cDNA from RNA or ssDNA in only 30 min
– High yields of first-strand cDNA
– No intrinsic RNase H activity
– Thermostable – working temperature range 37-50 ºC

Applications: 
– First-strand cDNA synthesis for RT-PCR and RT-qPCR
– Reverse transcription at elevated temperatures (ideal for high GC-content templates)
– Synthesis of cDNA for cloning and expression
– Analysis of RNA by primer extension

High sensitivity of NZY Reverse Transcriptase:
The high sensitivity of NZY Reverse Transcriptase is demonstrated using decreasing amounts of total RNA isolated from mouse liver (1 μg – 10 pg) as starting material in a 20 μL first-strand cDNA synthesis reaction with 200 units of the enzyme.

NZY Reverse Transcriptase_Figure1

After the reverse transcription, 1 μL of the resultant cDNA was used as a template to amplify the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in a 25 μL end-point PCR reaction with Supreme NZYTaq II DNA polymerase. NC: No template control. Lane M: NZYDNA Ladder III

Specifications:

Product length:up to 7 kb
Sensitivity:high
Optimal reaction temperature: 50 ºC
Available as Kits:NZY First-Strand cDNA Synthesis Kit (MB125); NZY First-Strand cDNA Synthesis Kit, separate oligos (MB170)
Speed:30 min
Denaturing conditions:NZY Reverse Transcriptase is inhibited in the presence of metal chelators (e.g. EDTA), inorganic phosphate, pyrophosphate and polyamines. The enzyme is inactivated at 85 °C for 5 min
Lyo format available:Lyo NZY Reverse Transcriptase (MB409)
Storage conditions:Store at -20 ºC
Shipping conditions:Shipped with dry ice

 

Components:
– NZY Reverse Transcriptase (200 U/μL)
– Reaction Buffer (10x)

 

NZYTech Reverse Transcriptases: Selection Guide

NZYRT-Guide

1. NZY M-MuLV First-Strand cDNA Synthesis Kit
2. NZY M-MuLV First-Strand cDNA Synthesis Kit, separate oligos

Product Brochure EN
MSDS PT
MSDS EN
Certificate of Analysis ZN031
Certificate of Analysis NM071
Certificate of Analysis NM041
Certificate of Analysis NM021
Certificate of Analysis 19051

NZY ReverNZY Reverse Transcriptase_Figure2se Transcriptase vs the competition. A 10-fold serial dilution of total RNA of mouse liver (1 μg to 1 ng) was reverse transcribed using 200 units of NZY Reverse Transcriptase or a popular reverse transcriptase. The resultant cDNA was then used as template in a PCR using specific primers for amplification of the mouse GAPDH gene with Supreme NZYTaq II DNA polymerase.  NC: No template control. Lane M: NZYDNA Ladder III.

 

 

 

 

 

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. Is the NZYReverse Transcriptase RNase H minus?
The enzyme has no intrinsic RNase H activity.

3. The reaction buffer in my NZYReverse Transcriptase has precipitated. Can I still use?
The NZYReverse Transcriptase 10x Reaction Buffer can on occasions form a white precipitate after repeated freeze/thaws. If this happens, mix thoroughly to resuspend the precipitate. If you verify non-conformity of results, then the buffer needs to be replaced.

4. 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.

5. 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.

6. 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. Ensure the use of NZY Ribonuclease Inhibitor: addition of this inhibitor is essential when using less than 50 ng of RNA in order to safeguard the template against degradation due to ribonuclease 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.

7. 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.

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