Introduction

Hipure Microbial RNA kit is suitable for extracting high-purity total RNA from bacterial/yeast culture medium. This kit combines high-efficiency Magzol Reagent (one-step extraction reagent) and silicagel column purification technology to complete the extraction of high-purity total RNA in only 40 minutes. The obtained RNA can be directly used in RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from bacteria, yeast cells
Applications	RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Bacteria, yeast cells
Sample amount	Bacteria: <10^9；Yeast cells：<2 x10^7
Elution volume	≥30μl
Time per run	≤40 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.

Advantages

• Fast – several samples can be extracted in 30 minutes
• High purity – the purified RNA can be directly used in various downstream applications
• High recovery – RNA can be recovered at the level of PG
• Good repeatability – silica gel column purification technology can obtain ideal results every time
• Broad spectrum – it can deal with various bacteria, including Gram-positive bacteria that are difficult to be lysed
• Sufficient components – the kit contains carried wall breaking enzymes and glass beads

Kit Contents

Contents	R418202	R418203
Purification Times	50 Preps	250 Preps
HiPure RNA Mini Columns	50	250
2ml Collection Tubes	50	250
Glass Beads (0.1-0.6mm)	30 g	150 g
DNase I	600 μl	5 x 600 μl
DNase Buffer	6 ml	30 ml
Protease Dissolve Buffer	1.8 ml	15 ml
Buffer ATL	50 ml	200 ml
Buffer PHC	50 ml	200 ml
Buffer GXP2*	20 ml	100 ml
Buffer RW1	50 ml	250 ml
Buffer RW2*	20 ml	2 x 50 ml
RNase Free Water	10 ml	30 ml

Storage and Stability

Buffer PHC should be stored at 2–8°C upon arrival. DNase I should be stored at -20°C. However, short-term storage (DNase I up to 1 weeks, Buffer PHC up to 12 weeks) at room temperature(15–25°C) does not affect their performance. The remaining kit components can be stored at roomtemperature (15–25°C) and are stable for at least 18 months under these conditions.

Hipure Microbial RNA kit is suitable for extracting high-purity total RNA from bacterial/yeast culture medium. This kit combines high-efficiency Magzol Reagent (one-step extraction reagent) and silicagel column purification technology to complete the extraction of high-purity total RNA in only 40 minutes. The obtained RNA can be directly used in RT-PCR, Northern blot, poly-A + purification, nucleic acid protection and in vitro translation.

Overview

• Isolate all sizes of circulating and exosomal RNA, including microRNA
• Versatile urine input ranges
• No phenol extractions
• No carrier RNA
• Bind and elute all RNA irrespective of size or GC content, without bias
• Concentrate circulating RNA and exosomal RNA into flexible elution volumes
• Purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen’s NGS services
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a fast, reliable and convenient method to purify and concentrate high quality, high purity and inhibitor-free cell-free circulating RNA, including exosomal RNA as well as viral RNA from fresh, preserved or frozen urine samples. All components for the purification are provided in one convenient and fast kit for the easy processing of small input volumes of bodily fluids. The purified urine RNA is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, expression array assays, and NGS.

Background

Recent evidence indicates that cell-free circulating RNA (cf-RNA) including exosomal RNA in urine contains valuable information for the discovery of biomarkers that can help for the early detection of certain cancer types and for monitoring the disease status as well as for the detection of any infectious pathogens. Exosomes can be found in saliva, blood, urine, amniotic fluid and malignant ascitic fluids, among other biological fluids. Evidence has been accumulating recently that these vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours, tissue repair, neural communication and transfer of pathogenic proteins. For this reason exosomal RNAs may serve as biomarkers for various diseases including cancer. The advantage for using urine as a source for cancer biomarkers is that it can be acquired in large quantities without using invasive procedures. In addition, repeated sampling from the same individual is applicable, which facilitates longitudinal studies. There are many advantages favouring the use of urinary nucleic acid for cancer biomarker discovery over blood, tissue samples or other bodily fluids, including: (1) urine is non-infectious for HIV and less infectious for many other pathogens; (2) the profile of urinary nucleic acid is similar to that in plasma or serum but with a lower concentration; (3) Nucleic acid purification from urine is technically much easier because of its low protein concentration (1000-fold lower than blood).

Urine Cell-Free Circulating RNA Purification Mini Kit

For sample volumes ranging from 250 µL to 2 mL.

Urine Cell-Free Circulating RNA Purification Midi Kit

For sample volumes ranging from 2 mL to 10 mL. The first column will handle the large volume input of urine that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL.

Urine Cell-Free Circulating RNA Purification Maxi Kit

For sample volumes ranging from 10 mL to 30 mL. The first column will handle the large volume input of urine that is followed by a concentration on a mini column for a final elution of 50 µL to 100 µL

Details

Supporting Data

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Kit Specifications
Minimum Urine Input	10 mL
Maximum Urine Input	30 mL
Size of RNA Purified	All sizes including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	40-45 minutes
Average Yields	Variable depending on specimen

*Please check page 6 of the product insert for average yields and common RNA quantification methods.

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. These kits are stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Component	Cat. 56900 (50 preps)	Cat. 57000 (20 preps)	Cat. 57100 (10 preps)
Binding Solution K	25 mL	75 mL	1 x 75 mL 1 x 25 mL
Lysis Buffer A	30 mL	20 mL	20 mL
Wash Solution A	18 mL	18 mL	18 mL
Elution Solution A	6 mL	6 mL	6 mL
Mini Spin Columns	50	20	10
Midi Spin Columns	–	20	–
Maxi Spin Columns	–	–	10
Collection Tubes	50	20	10
Elution Tubes (1.7 mL)	50	20	10
Product Insert	1	1	1

Bis-Sulfone-PEG9-DBCO is a bis-alkylating labeling reagent that is selective for the cysteine sulfur atoms from a native disulfide. These reagents undergo bis-alkylation to conjugate both thiols derived from the two cysteine residues of a reduced native disulfide bond such as the interchain disulfide bonds of an antibody. The reaction results in covalent rebridging of the disulfide bond via a three carbon bridge leaving the protein structurally intact. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Bis-Sulfone-PEG9-DBCO is a bis-alkylating labeling reagent that is selective for the cysteine sulfur atoms from a native disulfide. These reagents undergo bis-alkylation to conjugate both thiols derived from the two cysteine residues of a reduced native disulfide bond such as the interchain disulfide bonds of an antibody. The reaction results in covalent rebridging of the disulfide bond via a three carbon bridge leaving the protein structurally intact. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.