aRNA Synthesis Kit

Products - Amplify & Label - aRNA Synthesis Kits

For amplification of RNA for microarray, splice variant microarrays and other down stream applications where high quality aRNA is required.

This kit is optimized to use 50ng-1µg of total cellular RNA as starting material. The process includes the following steps:

First Strand cDNA Synthesis
Second Strand cDNA Synthesis
Amplification of ds cDNA
Purification of ds cDNA
In Vitro Transcription with T7 RNA Polymerase featuring T7 Booster, a proprietary blend of enhancers to augment the polymerase activity resulting in an increase in both the yield and the amount of full-length transcripts. Special proteins are added into this enhancer mix that protects the nascent RNA strand during synthesis.
RNA Purification

If used properly this kit will generate 5-250µg of amplified anti-sense mRNA (aRNA) ready for labeling.

Table of Contents

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your genomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use ArrayIt^® aRNA Synthesis kit.

Quality Control
TeleChem assures the performance of this product. The finest scientific research went into the development of this product. Rigorous quality control monitoring on a lot-by-lot basis guarantees that the ingredients conform to the highest industry standards.

Product Advantages

The most comprehensive kit available
Robust RT reaction; allows full length cDNA synthesis with unique primers
Unique second strand synthesis: takes the advantage of both linear and exponential amplifications
Purification using unique columns that minimize loss
Optimized reactions with unique additives, resulting in a huge incremental yield

Contents

Item

Packaging

Storage

Enzyme Mix 1

16.5µl in 0.5ml tube

-20^oC Freezer

Enhancer solution

50µl in 0.5ml tube

-20^oC Freezer

Reaction buffer 1

50µl in 0.5ml tube

-20^oC Freezer

dNTP Mix

22µl in 0.5ml tube

-20^oC Freezer

DTT

50µl in 0.5ml tube

-20^oC Freezer

Primer 1AS

11µl in 0.5ml tube

-20^oC Freezer

Primer 2AS

11µl in 0.5ml tube

-20^oC Freezer

Primer 3AS

22µl in 0.5ml tube

-20^oC Freezer

Primer 4AS

11µl in 0.5ml tube

-20^oC Freezer

Enzyme Mix 2

11µl in 0.5ml tube

-20^oC Freezer

Reaction Buffer 2

150µl in 0.5ml tube

-20^oC Freezer

DNA Binding Buffer

1.1ml in 2ml tube

Room Temperature

DNA Wash Buffer Concentrate

0.5ml in 8ml bottle

Room Temperature

DNA Micro Column (without cap)

10

Room Temperature

Elution Tube (1.5ml)

10

Room Temperature

2 ml Wash Tube

10

Room Temperature

Nuclease Free Water

1.5ml in 2ml tube

-20^oC Freezer

Enzyme Mix 3

22µl in 0.5ml tube

-20^oC Freezer

NTP Mix

79.2µl in 0.5ml tube

-20^oC Freezer

Reaction Buffer 3

22µl in 0.5ml tube

-20^oC Freezer

DNAse

11µl in 0.5ml tube

-20^oC Freezer

Sodium Acetate Solution

50µl in 0.5ml tube

-20^oC Freezer

Linear Acrylamide

11µl in 0.5ml tube

-20^oC Freezer

Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance. By electronic mail: arrayit@arrayit.com (under the subject heading please type ArrayIt technical assistance). By telephone: (408) 744-1331, Monday—Friday PST 9:00am - 4:30pm. Please remember that we want to hear about your successes!

Requirements
Items needed but not supplied

Ethanol 100%
Ethanol 70%
Ice for incubation
PCR tubes

Equipment needed

Thermal cycler
Refrigerated microfuge capable of 10,000 rpm or more
Micro-pipettes
Vortex mixer
Homogenizer (if using tissue)
Temperature regulated water bath or dry bath

Note: Spin all tubes briefly before opening.

This kit is optimized to use 50ng-1µg of total cellular RNA as starting material. The process includes the following steps:

First Strand cDNA Synthesis
Second Strand cDNA Synthesis
Amplification of ds cDNA
Purification of ds cDNA
In Vitro Transcription with T7 RNA Polymerase
RNA Purification

If used properly this kit will generate 5-250µg of amplified antisense mRNA (aRNA) ready for labeling.

Total working time required for this kit is between 5-6 hrs.

Short Protocol
1. First Strand cDNA Synthesis.
2. Second Strand cDNA Synthesis.
3. Amplification of dsDNA.
4. Purification of dsDNA.
5. In Vitro Transcription with T7 RNA Polymerase.
6. RNA Purification.

Complete Protocol
Step 1. First Strand cDNA Synthesis.
In a PCR tube on ice add the following:

Eluted RNA (50ng-1g) x µl
Primer 1AS 1µl
Nuclease Free Water to make up 11.5µl

Incubate the reaction tube at 65^oC for 5 mins.
Incubate at room temperature for 10mins to allow the annealing of the primers.
Add the following to the reaction tube:

Enhancer Solution 2µl
Reaction Buffer 1 2µl
Enzyme Mix 1 1.5µl
dNTP Mix 1µl
Primer 2AS 1µl
DTT 1µl

Total reaction volume 20µl

Incubate in a PCR machine at 42^oC for 60 mins.
Followed by 95^oC for 5mins to inactivate the Reverse Transcriptase.
Chill on ice for 5mins and briefly spin to collect all the liquid.

Step 2. Second Strand cDNA Synthesis.
To the reaction tube on ice add the following:

Enzyme Mix 2 1µl
Reaction Buffer 2 10 µl
dNTP Mix 1 µl
Primer 3AS 2 µl
Nuclease free water 16 µl
Total Reaction Volume 50 µl

Perform the following pattern of incubation in a thermal cycler:

Incubation 37^oC for 10 mins
Denaturation 98^oC for 40 secs
Anneal 60^oC for 30 secs
Extend 72^oC for 4 mins
Incubation 4^oC for 5 mins

Step 3. Amplification of ds cDNA.
Briefly spin to collect all the liquid and add 1 µl of Primer 4 AS.
Perform five to ten cycles of PCR in a thermal cycler depending on the starting amount of RNA viz. when starting with 50 ng total RNA perform 10 cycles, when amplifying from 1 µg total RNA perform 5 cycles.

Initial Denaturation 98^oC for 2 mins
Denaturation 98^oC for 30 secs (Cycle 5-10 times)
Anneal 60^oC for 30 secs (Cycle 5-10 times)
Extend 72^oC for 4 mins (Cycle 5-10 times)
Terminal Extension 72^oC for 10 mins
Incubation 4^oC for 5 mins

Briefly spin to collect all the liquid.

Step 4. Purification of ds cDNA.

Before starting add 2 ml of 100% ethanol to DNA Wash Buffer Concentrate
Add 100 µl DNA Binding Buffer
Load the entire contents onto a DNA micro column and place the column in a 2 ml wash tube
Centrifuge for 1 min at full speed in a microfuge (>10000 rpm), discard the flow through and reuse the wash tube
Add 100 µl of DNA wash buffer to the column and spin at full speed for 1 min
Add another 100 µl of DNA wash buffer to the column and spin at full speed for 2 min
Discard the 2 ml wash tube and carefully place the column in an elution tube
Add 8 µl of nuclease free water that has been pre-warmed to 60^oC to the center of the column
Wait for 2 mins and spin at full speed for 1 min
Reload the filtrate onto the column
Wait for 2 mins and spin at full speed for 1 min
Discard the column and use the cDNA in the next step

Note: If desired the eluted cDNA can now be frozen and stored at –20^oC.

Step 5. In Vitro Transcription with T7 RNA Polymerase.
Place a PCR tube at room temperature and add the following:
Note: Assemble the T7 reaction at room temperature, if the reaction buffer 3 shows a white precipitate; incubate the buffer at 37^oC for 5 mins and allow to cool to room temperature before setting up the reaction

Purified cDNA 6.8 µl
Reaction Buffer 3 2 µl
NTP Mix 7.2 µl
DTT 2 µl
Enzyme Mix 3 2 µl
Total reaction volume 20 µl

Incubate in a PCR machine at 37^oC for 30-60 mins.
Use 30 mins when the starting total RNA is 1µg and 60 mins when the starting total RNA is 50 ng.
Chill on ice for 5 mins and briefly spin to collect all the liquid.

Step 6. RNA Purification.

Add 1 µl of DNAse to the reaction tube and incubate 37^oC for 15 mins
Chill on ice for 5 mins and briefly spin to collect all the liquid
Add 2 µl of Sodium Acetate Solution
Add 50 µl 100% ethanol
Add 1 µl of Linear Acrylamide
Mix gently with a pipette tip and incubate at -20^oC for 30 mins
Centrifuge for 15 mins at full speed in a microfuge (>10000 rpm) at 4^oC
Remove the supernatant carefully and gently rinse the pellet twice with 200 µl of 70% ethanol
Briefly air dry the pellet and reconstitute in 25 µl nuclease free water
RNA can be stored at -70^oC as a dry pellet or re-suspended in nuclease free water or TE or other suitable buffer

Ordering Information

Product

Description

Catalog ID

Price per kit (US dollars)*

aRNA Synthesis Amplification Kit(10-reactions)

This kit is optimized to use 50ng-1µg of total cellular RNA as starting material. If used properly this kit will generate 5-250µg of amplified antisense mRNA (aRNA) ready for labeling.

ASK

$799.00

*To order ArrayIt^® Brand Products: call 408-744-1331, fax 408-744-1711, email arrayit@arrayit.com or click on the button above to proceed directly to the purchase page.

*International pricing may vary as much as 30% (or more depending on country) due to import duties, stocking fees and technical support.

Warranty
ArrayIt® brand products have been developed scientifically, and are sold for research and development purposes. Extreme care and exact attention should be practiced in the use of the materials described herein. All ArrayIt® brand products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with ArrayIt® products should be reported to TeleChem immediately. TeleChem's liability is limited to the replacement of the product, or a full refund. Any misuse of this product is the full responsibility of the user, and TeleChem makes no warranty or guarantee under such circumstances.