Tissue RNA kit

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Tissue RNA Kit

Tissue RNA Kit

Tissue RNA extraction and purification kit. Compared to other purification method in the market, this kit is simplified with only one lysis buffer and one wash buffer. The purified RNA is ready for Northern analysis, RT-PCR, MicroArray and real-time PCR. It is especially suitable for quantitative gene expression study.

Features:

Fast and reliable: the whole procedure can be completed in 20 minutes.

Stable RNA in DEPC water.

No toxic organic chemicals.

No genomic DNA contaminations.

96-well Tissue RNA kit Cat#: R6811-01 Size: 4x96 96-well Tissue/Eukaryote Total RNA Purification Kit Learn More $ 780.00 Add to Wishlist Add to Compare	Tissue RNA midi kit Cat#: R6312-01 Size: 10 Purification of total RNA up to 1 mg from 110^8?eukaryotes or ~ 200 mg tissue specimen Learn More $ 80.00 Add to Wishlist Add to Compare	Tissue RNA mini kit Cat#: R6311-02 Size: 250 Purification of total RNA up to 100 microgram from 510^6 eukaryotes or 30 mg tissue specimen. Learn More $ 650.00 Add to Wishlist Add to Compare	Tissue RNA mini kit Cat#: R6311-01 Size: 50 Tissue RNA extraction purification mini kit purifies total RNA up to 100 microgram from 510^6 eukaryotes or 30 mg tissue specimen. Learn More $ 148.00 Add to Wishlist Add to Compare
Tissue RNA maxi kit Cat#: R6314-02 Size: 25 Purification of total RNA up to 5 mg from 5 10^8?eukaryotes or ~ 1 g tissue specimen Learn More $ 270.00 Add to Wishlist Add to Compare	Tissue RNA maxi kit Cat#: R6314-01 Size: 10 Purification of total RNA up to 5 mg from 5 10^8?eukaryotes or ~ 1 g tissue specimen Learn More $ 120.00 Add to Wishlist Add to Compare	Tissue RNA Mega kit Cat#: R6315-02 Size: 10 Purification of total RNA up to 15 mg from 2 10^9?eukaryotes or ~ 5 g tissue specimen Learn More $ 580.00 Add to Wishlist Add to Compare	Tissue RNA Mega kit Cat#: R6315-01 Size: 2 Purification of total RNA up to 15 mg from 2 10^9?eukaryotes or ~ 5 g tissue specimen Learn More $ 120.00 Add to Wishlist Add to Compare

Introduction

The EZgeneTM Tissue RNA kit provides an easy and fast method for isolating total RNA from tissues, cultured cells within 30 min. Only trace genomic DNA exists in the purified RNA, which can be eliminated by DNase I treatment (See detail in the protocol) when it is necessary.

Storage and Stability

DNase I (optional) should be stored at -20℃. All other components can be stored at room temperature. All kit components are guaranteed for 1 year from the date of purchasing.

Kit Contents

Catalog#

R6311-01

R6311-02

Buffer LY

28 mL

130 mL

RNA Wash Buffer *

24 mL

112 mL

DEPC-Treated ddH2O

10 mL

30 mL

DNase Stop Buffer

4.8 mL(included)

24 mL(included)

ezBind Columns

50

250

Collection Tubes

100

500

User Menu

1

1

Add 36 mL or 168 mL 100% ethanol into corresponding wash buffer before use.

Before Starting

Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps.

Important

R Add 1% volume of b-mercaptoethanol to Buffer LY before use and store at 4℃.

R Add 36 mL (R6311-01) or 168 mL (R6311-02) 100% ethanol to RNA Wash Buffer before use.

R All centrifugation steps should be carried out at room temperature.

R Add 7.2 mL (R6311-01) or 36 mL (R6311-02) 100% ethanol to DNase Stop Buffer before use.

Materials supplied by users

R Tabletop microcentrifuge and 1.5 mL sterile tubes.

R Vacuum manifold if use vacuum protocol.

R 100% ethanol

R Optional: DNase I, DNase Buffer

Note: Perform all steps including centrifugation at room temperature

Protocol For Extracting Total RNA From Cultured Cells

1. Cell preparation:

a. For total RNA extraction from suspension cultured cells, collect cells by centrifuging at 300 x g for 5 min. Wash the cell pellet by 4 ℃1 x PBS and centrifuge at 300 x g for 5 min. Discard the supernatant.

b. For total RNA extraction from adherent cultured cells, remove the culture medium. Wash the cell by 4 ℃ 1xPBS and remove the PBS. Or following the regular protocol to collect the cells.

2. Add 500 μL Buffer LY to the cell pellet or directly into the well (for adherent cells) according to the Table 1.

Ensure that b-mercaptoethanol has been added before use.

3. Homogenize the lysate by vortexing vigorously or repeated pipetting. If the solution is clear, go to step 5, otherwise go to step 4.

4. Centrifuge the solution at top speed for 2 min and transfer the clear lysate to a clean 1.5 mL tube.

5. Add 1/2 volume 100% ethanol into the lysate (for example: 250 μL 100% ethanol for 500 μL lysate) and pipet 5 times to mix the solution. Vortex briefly if any precipitations.

6. Transfer the solution into the binding column and centrifuge at 14,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.

7. Add 400 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

Ensure that ethanol has been added to RNA Wash Buffer before use.

8. Optional: Add 50 μL DNase I (5U，RNase-free) solution onto the middle of the column and incubate at room temperature for 15 min. Add 200 μL DNase Stop Buffer onto the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through. Add 300 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

9. Add another 400 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 30 s. Discard the flow-through.

10. Centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

11. Centrifuge at 14,000 rpm for another 1 min.

It is critical to remove residual ethanol for optimal elution.

12. Place the column to a RNase-free 1.5 mL tube and add 50-100 μL DEPC-treated water to the column and centrifuge at 14,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution at -20℃.

Protocol For Extracting Total RNA From Animal Tissue

1. Quickly weigh an appropriate tissue mass according to Table 1 (Page 12) and transfer the tissue into a 1.5 mL tube containing 500 μL Buffer LY and homogenize the tissue by a rotor starter or an ultrasonic homogenizer on ice.

Ensure that b-mercaptoethanol has been added before use. Use of too much tissue per preparation will cause genomic DNA contamination.

2. Centrifuge the lysate for 10 min at 14,000 rpm at room temperature and transfer the cleared lysate to a clean 1.5 mL tube.

3. Add 0.5 volume 100% ethanol to the lysate (for example: 250 μL 100% ethanol for 500 μL lysate).

4. Transfer the solution into the binding column and centrifuge at 14,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.

5. Add 400 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

Ensure that ethanol has been added to RNA Wash Buffer before use.

6. Optional: Add 50 μL DNase I (5U，RNase-free) solution onto the middle of the column and incubate at room temperature for 15 min. Then add 200 μL DNase Stop Buffer into the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through. Add 300 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

7. Add another 400 μL RNA Wash Buffer to the column and centrifuge at 14,000 rpm for 30 s. Discard the flow-through.

8. Centrifuge at 14,000 rpm for 1 min. Discard the flow-through.

9. Centrifuge at 14,000 rpm for another 1 min.

It is critical to remove residual ethanol for optimal elution.

10. Place the column onto a RNase-free 1.5 mL tube, add 50-100 μL DEPC-treated water to the column and centrifuge at 14,000 rpm for 2 min. The RNA is in the flow-through liquid. Store the RNA solution at -20 ℃.

Table 1. Recommended Amounts of Tissue and Cells for Preparation

Sample	Max Tissue or Cell Mass/500 mL Buffer LY	Max Tissue or Cell Mass per Prep	Total RNA Yield (mg)
Liver	80 mg	30 mg	130
Kidney	50 mg	20 mg	45
Muscle	80 mg	80 mg	50
Spleen	40 mg	15 mg	80
Heart	160 mg	100 mg	50
Brain	160 mg	120 mg	80
Lung	160 mg	100 mg	70
Pancreas	80 mg	30 mg	100
Tomato Leaves	100 mg	200 mg	30
HeLa Cells	5x106	5x106	120

Note: It is highly recommended that RNA quality be determined before downstream applications. The quality of RNA can be assessed by denatured agarose gel electrophoresis with the ethidium bromide staining. Several sharp bands should appear on the gel including 28S and 18S ribosomal RNA bands as well as certain populations of mRNA and bands. If these bands smear towards lower molecular weight RNAs, then the RNA has undergone major degradation during preparation, handling or storage, RNA molecule less than 200 bases in length do not efficiently bind to the RNA column. An A260/A280 ratio of 1.8-2.0 corresponds to 90-100% pure nucleic acid.

Trouble Shooting Guide

Problem

Possible reason

Suggested Improvement

Low A260/A280 ratios

Protein contamination

Do a Phenol:Chloroform extraction. Loss of total RNA (up to 40%) should be expected.

Low A260/A280 ratios

Guanidine Thiocyanate contamination

Add 2.5 volumes of ethanol and 0.1M NaCl (final concentration) to precipitate RNA. Incubate for 30 min at -20℃. Centrifuge at 10,000 g for 15 min at 4°C. Resuspend the RNA pellet in DEPC-treated water.

Low Yield

RNA in sample degraded

Freeze samples immediately in liquid nitrogen and store at -70℃ after collect it.

Low Yield

The binding capacity of the membrane in the spin column was exceeded

Use of too much tissue sample exceeding the binding capacity of spin column will cause the decreasing of total RNA yield.

Low Yield

Ethanol not added to buffer

Add ethanol to the RNA Wash Buffer and DNase Stop Solution before purification.

Genomic DNA contamination

Too much total RNA sample was used in RT-PCR.

Reduce total RNA amount used in RT-PCR to 50-100 ng.

Genomic DNA contamination

The sample may contain too much genomic DNA.

Reduce the amount of starting tissue in the preparation of the homogenate. Most tissues will not show a genomic DNA contamination problem at 30 mg or less per prep. Reduce cell numbers to 1-2x106 or increase buffer volume and do multiple loadings to column.

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Catalog#	R6311-01	R6311-02
Buffer LY	28 mL	130 mL
RNA Wash Buffer *	24 mL	112 mL
DEPC-Treated ddH2O	10 mL	30 mL
DNase Stop Buffer	4.8 mL(included)	24 mL(included)
ezBind Columns	50	250
Collection Tubes	100	500
User Menu	1	1

Problem	Possible reason	Suggested Improvement
Low A260/A280 ratios	Protein contamination	Do a Phenol:Chloroform extraction. Loss of total RNA (up to 40%) should be expected.
Low A260/A280 ratios	Guanidine Thiocyanate contamination	Add 2.5 volumes of ethanol and 0.1M NaCl (final concentration) to precipitate RNA. Incubate for 30 min at -20℃. Centrifuge at 10,000 g for 15 min at 4°C. Resuspend the RNA pellet in DEPC-treated water.
Low Yield	RNA in sample degraded	Freeze samples immediately in liquid nitrogen and store at -70℃ after collect it.
Low Yield	The binding capacity of the membrane in the spin column was exceeded	Use of too much tissue sample exceeding the binding capacity of spin column will cause the decreasing of total RNA yield.
Low Yield	Ethanol not added to buffer	Add ethanol to the RNA Wash Buffer and DNase Stop Solution before purification.
Genomic DNA contamination	Too much total RNA sample was used in RT-PCR.	Reduce total RNA amount used in RT-PCR to 50-100 ng.
Genomic DNA contamination	The sample may contain too much genomic DNA.	Reduce the amount of starting tissue in the preparation of the homogenate. Most tissues will not show a genomic DNA contamination problem at 30 mg or less per prep. Reduce cell numbers to 1-2x106 or increase buffer volume and do multiple loadings to column.

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