Introduction

Hipure viral RNA kit is suitable for purifying viral RNA from samples such as cell-free body fluid or culture medium. The kit is based on silica gel column purification technology. It requires no toxic phenol chloroform extraction and time-consuming alcohol precipitation in the extraction. The whole extraction process takes only 25 minutes. The kit is suitable for extracting viral RNA from 1-140µl cell-free fluid samples such as serum, plasma, cell-free body fluid or culture medium. The product has successfully extracted hepatitis B A/C, hepatitis C RNA, SARS and HIV. The obtained RNA can be directly used for RT-PCR, Northern hybridization and virus detection.

Details

Specifications

Features	Specifications
Main Functions	Extract viral RNA from 140μl cell-free samples
Applications	RT-PCR, Northern hybridization, and various virus detection
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Cell-free body fluid or culture medium
Sample amount	140μl
Elution volume	≥15μ
Time per run	≤25 minutes
Liquid carrying volume per column	800μl
Binding yield of column	100μg

Principle

Hipure silica gel column is based on glass fiber filter membrane with high binding force. Under the condition of high concentration of ionizing agent (such as guanidine hydrochloride or guanidine isothiocyanate), the filter membrane can adsorb nucleic acid through hydrogen bond and electrostatic, while protein and other impurities are not adsorbed and removed. The filter membrane adsorbed with nucleic acid is washed to remove the residual protein and salt. Finally, the nucleic acid adsorbed on the filter membrane can be washed out with low salt buffer (such as buffer TE) or water. The obtained nucleic acid has high purity and can be directly used in various downstream experiments.

The sample is homogenized and lysed in the lysate, and RNA is released into the lysate. The high concentration of guanidine isothiocyanate contained in the lysate denatured and inactivated endogenous or exogenous RNase, while RNA is protected from degradation. The lysate is centrifuged to remove insoluble impurities. After adding ethanol to adjust the binding conditions, it is transferred to the column for filtration. RNA is adsorbed on the membrane of the column, while protein is removed without adsorption. The column is washed with buffer VHB to remove protein and other impurities, washed with buffer RW2 to remove salt. Finally the RNA is eluted by RNase free water. The eluted RNA can be directly used in RT-PCR, Northern blot, poly-A purification, in vitro translation, etc.

Advantages

• Fast – several samples can be extracted in 20 minutes by column method
• High quality – high purity total RNA can be directly used in various sensitive downstream applications
• Safe – no phenol chloroform extraction required
• Sensitive – RNA can be recovered at the level of PG
• High yield – carrier RNA contained in the product maximize the recovery of trace nucleic acid

Kit Contents

Contents	R417102	R417103
Purification Times	50 Preps	250 Preps
HiPure Viral Micro Columns	50	250
2ml Collection Tubes	100	500
Buffer VRL	50 ml	200 ml
Carrier RNA	310 µg	3 x 310 µg
Buffer VHB*	13 ml	110 ml
Buffer RW2*	20 ml	2 x 50 ml
Nuclease Free Water	10 ml	30 ml

Storage and Stability

Carrier RNA should be stored at 2–8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15–25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15–25°C) and are stable for at least 18 months under theseconditions

Hipure viral RNA kit is suitable for purifying viral RNA from samples such as cell-free body fluid or culture medium. The kit is based on silica gel column purification technology. It requires no toxic phenol chloroform extraction and time-consuming alcohol precipitation in the extraction. The whole extraction process takes only 25 minutes. The kit is suitable for extracting viral RNA from 1-140µl cell-free fluid samples such as serum, plasma, cell-free body fluid or culture medium. The product has successfully extracted hepatitis B A/C, hepatitis C RNA, SARS and HIV. The obtained RNA can be directly used for RT-PCR, Northern hybridization and virus detection.

Magnetic Beads (tRNA Purification)

Solid Phase Reversible Immobilization magnetic beads has been used for nucleic acids purification because they are effective, simple, and fast. The carboxyl groups coated beads are paramagnetic particles that reversibly bind to nucleic acid. However, there is a drawback for magnetic beads that it can only purify DNA/RNA that are 100 base pairs or longer. tRNA and other DNA/RNA fragments shorter than 100 base pairs are not effectively recovered. We have developed Magnetic Beads (tRNA Purification) to overcome the hurdle.

The Magnetic Beads (tRNA Purification) solves the tRNA and short DNA/RNA purification problem. The beads with our proprietary technology purify tRNA effectively by removing impurities and unwanted components such as dNTPs, detergents, salts, proteins, and other contaminants. The magnetic bead reagents can be used for oligo (>70 nt) applications.

Workflow without large RNA/DNA contamination

In the case of samples contaminated with RNA/DNA such as rRNA and DNA, our magnetic beads can effectively remove RNA/DNA that are 180 nt and larger. Purified tRNA are ideal for applications requiring high quality, as the fragments are free of impurities and contaminants.

Workflow with large RNA/DNA contamination

Comparison of tRNA and 70 nt oligos recovery. BioDynami beads (tRNA purification) successfully recovered DNA fragments both yeast tRNA and 70 nt oligos.

Recovery of tRNA and 70 nt oligos with BioDynami magnetic Beads (tRNA Purification). Yeast tRNA and 70 nt oligos were used as input. Input and recovered oligos were quantified with ssDNA Quantification kit (BioDynami Cat. # 40043) and RNA Quantification kit (BioDynami Cat. # 40044).

Depletion of larger RNAs. Left panel: depletion of 28S rRNA and 18S rRNA. Right panel: depletion of RNA of 180 nt and larger.

Features

Removal of unwanted components and other impurities

Purification of tRNA and oligos (>70 nt)

tRNA

RNA fragments 70 nt or longer

DNA/RNA hybrid fragments 70 nt or longer

Oligo and chimeric oligo 70 nt or longer

dsDNA fragments 70 bp or longer

ssDNA fragments 70 nt or longer

Removal of larger RNA/DNA contamination:

18S rRNA

28S rRNA

RNA/DNA> 180 nt

Solid Phase Reversible Immobilization magnetic beads has been used for nucleic acids purification because they are effective, simple, and fast. The carboxyl groups coated beads are paramagnetic particles that reversibly bind to nucleic acid. However, there is a drawback for magnetic beads that it can only purify DNA/RNA that are 100 base pairs or longer. tRNA and other DNA/RNA fragments shorter than 100 base pairs are not effectively recovered. We have developed Magnetic Beads (tRNA Purification) to overcome the hurdle.

Overview

• Convenient ready-to-use solution
• High sensitivity and yield
• Robust amplification
• Available in 3 convenient sizes: 100, 200 and 500 reactions (20 µL vol)
• This product is also a recommended add-on to be used with Norgen’s COVID-19 Primer and Probe Solutions and COVID-19 Positive Control 

Norgen’s 2X One-Step RT-PCR Master Mix is a ready-to-use solution that contains components required for RT-PCR amplification of RNA templates. The mix includes M-MuLV reverse transcriptase, Taq DNA polymerase, dNTPs, reaction buffer, MgCl₂, KCl, and a PCR enhancer/stabilizer. The user needs only to add the template, the primer set and water to the Master Mix to set up the RT-PCR reaction. This convenient 2X One-Step RT-PCR Master Mix reduces the time required to set up PCR reactions and reduces the possibility of contamination, particularly when preparing large numbers of reactions. The optimized master mix allows for robust amplification of RNA templates with high yields of PCR products.

Taq DNA Polymerase is a highly thermostable DNA polymerase that possesses a 5´→ 3´ polymerase activity and a very low 5´→ 3´ exonuclease activity. The source of Taq included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned Taq DNA Polymerase gene from Thermus aquaticus YT-1. M-MuLV Reverse Transcriptase is an RNA-directed DNA polymerase that can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. The source of the Reverse Transcriptase included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned reverse transcriptase gene from M-MuLV.

Norgen’s 2X One-Step RT-PCR Master Mix is available in 3 convenient sizes:

Cat # 28113 – 100 reactions (sufficient for 100 reactions x 20 µL reaction volume)
Cat # 28114 – 200 reactions (sufficient for 200 reactions x 20 µL reaction volume)
Cat # 28115 – 500 reactions (sufficient for 500 reactions x 20 µL reaction volume)

Details

Supporting Data

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Reagents supplied – Cat # 28114

• 2X One-Step RT-PCR Master Mix (2 Vials, 100 Reactions each) – Sufficient reagent for 200 x 20 µL reactions

Storage Conditions and Product Stability
2X One-Step RT-PCR Master Mix should be stored at -20°C. For everyday use an aliquot can be stored at 4°C for up to 3 months. Repeated freeze-thaw cycles are not recommended. When stored at the proper temperature this reagent is stable for at least 1 year.