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Products - Amplification & Labeling - mRNA Amplification Kits for DNA Microarray Applications in Human, Plant and Animal Genome Analysis

RNA-amplification
Arrayit has developed the most robust, comprehensive and complete kits available for mRNA amplification. The ArrayIt MiniAmp mRNA amplification kit is optimized to use 50 ng-1 g of total cellular RNA as starting material, and is highly recommended for use with our H25K Whole Human Genome Chip.

The mRNA amplification process includes the following steps:

  • First Strand full length ds cDNA Synthesis: Featuring RT Booster, a proprietary ionic based enhancer solution that improves reverse transcriptase activity resulting in an increase in both the yield and the amount of full length cDNA products.
  • Linear and exponential second strand cDNA Synthesis.
  • Amplification of ds cDNA.
  • High yield cDNA Purification of ds cDNA.
  • High yield 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.
  • Amplified RNA Purification.
  • If used properly this kit will generate 5-50 g of amplified sense strand mRNA ready for labeling with amino allyl dUTP based labeling.

Can be used stand alone or in conjunction with:

Table of Contents

  • Introduction
  • Quality Control
  • Product Advantages
  • Kit Contents
  • Technical Assistance
  • Requirements
  • Equipment Requirements
  • Short Protocol
  • Complete Protocol
  • Ordering Information
  • Warranty

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® MiniAmp mRNA amplification 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

  • Fully compatible with H25K Whole Human Genome Chips.
  • 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
  • cDNA purification using unique columns that minimize loss
  • Optimized T7 reaction with unique additives, resulting in a huge incremental yield

Kit Contents

Item

Packaging

Storage

Enzyme Mix 1

16.5 l in 0.5 ml tube

-20C Freezer

RT Booster (enhancer solution)

50 l in 0.5 ml tube

-20C Freezer

Reaction buffer 1

50 l in 0.5 ml tube

-20C Freezer

dNTP Mix

40 l in 0.5 ml tube

-20C Freezer

DTT

50 l in 0.5 ml tube

-20C Freezer

Primer 1 Mix

11 l in 0.5 ml tube

-20C Freezer

Primer 2

11 l in 0.5 ml tube

-20C Freezer

Primer 3

22 l in 0.5 ml tube

-20C Freezer

Primer 4

11 l in 0.5 ml tube

-20C Freezer

Enzyme Mix 2

11 l in 0.5 ml tube

-20C Freezer

Reaction Buffer 2

150 l in 0.5 ml tube

-20C Freezer

DNA Binding Buffer

1.1 ml in 2 ml tube

Room Temp

DNA Wash Buffer Concentrate

0.5 ml in 8 ml bottle

Room Temp

DNA Micro Column

10 each

Room Temp

Elution Tube (1.5 ml)

10 each

Room Temp

2 ml Wash Tube

10 each

Room Temp

Nuclease Free Water

1.5 ml in 2 ml tube

-20C Freezer

Enzyme Mix 3

22 l in 0.5 ml tube

-20C Freezer

NTP Mix

79.2 l in 0.5 ml tube

-20C Freezer

Reaction Buffer 3

22 l in 0.5 ml tube

-20C Freezer

DNAse

11 l in 0.5 ml tube

-20C Freezer

Sodium Acetate Solution

50 l in 0.5 ml tube

-20C Freezer

Linear Acrylamide

20 l in 0.5 ml tube

-20C 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
  • Micropipettors
  • Vortex mixer
  • Homogenizer (if using tissue)
  • Temperature regulated water bath or dry bath

IMPORTANT NOTE: Spin all tubes briefly before opening.

Short Protocol
This kit is optimized to use 50 ng-1 g of total cellular RNA as the starting material, and will generate 5-50 g of amplified sense strand mRNA suitable for labeling with aminoallyl dUTPs.

The process requires the following steps:
1. Perform first strand cDNA synthesis.
2. Perform second strand cDNA synthesis.
3. Amplify the double-stranded (ds) cDNA.
4. Purify the double-stranded cDNA.
5. Transcribe the ds cDNA with T7 RNA polymerase in vitro.
6. Purify the amplified RNA (aRNA).
7. Perform cDNA synthesis on aRNA using aminoallyl-labeled dNTPs.

Complete Protocol
1. Perform first strand cDNA synthesis.

To a PCR tube on ice, add the following:

  • Total cellular RNA (50 ng-1 g)  1-10 l
  • Primer 1 Mix                               1 l
  • Nuclease-free water                0-9 l
  • Total volume                           11 l

Incubate PCR tube at 65C for 5 min to denature the RNA.

Incubate PCR tube at 20-25C for 10 min to allow primer annealing.

Add the following to the PCR tube:

  • RT Booster (enhancer solution)   2 l
  • Reaction Buffer 1                    2 l
  • Enzyme Mix 1                         1.5 l
  • dNTP Mix                               1.5 l
  • Primer 2                                 1 l
  • DTT                                      1 l
  • Total reaction volume              20 l

Incubate in a PCR machine at 42C for 60 min.
Incubate at 95C for 5 min to inactivate the reverse transcriptase.
Chill on ice for 5 min.
Spin tube briefly to consolidate the 20 l sample.
Return tube to ice before proceeding to next step.

2. Perform second strand cDNA synthesis.
To the PCR tube on ice, add the following:

  • Enzyme mix 2                         1 l
  • Reaction buffer 2                    10 l
  • dNTP mix                               2.5 l
  • Primer 3                                 2 l
  • Nuclease-free water                14.5 l
  • Total reaction volume              50 l

Using a thermal cycler, perform the following steps:

  • Incubate at 37C for 10 min
  • Denature at 98C for 40 sec
  • Anneal at 60C for 30 sec
  • Extend at 72C for 4 min
  • Incubate at 4C for 5 min

3. Amplify the double-stranded (ds) cDNA.
Spin PCR tube briefly to consolidate the 50 l sample.
Add 1 l of Primer 4.
Using a thermal cycler, perform 5-10 cycles of PCR in a thermal cycler depending on the starting amount of RNA.  When starting with 50ng of total RNA or 2500 cells perform 10 cycles, when amplifying form 1 1 g of total RNA or 50,000 cells perform 5 cycles:

  • Initial Denaturation 98C for 2 min
  • Cycle 5-10 times using 98C for 30 sec, 60C for 30 sec, and 72C for 4 min
  • Extend 72C for 10 min
  • Incubate 4C for 5 min

Spin briefly to collect all the liquid.

4. Purify the double-stranded cDNA.
Note:  To prepare the DNA Wash Buffer, add 2 ml of 100% ethanol to the DNA Wash Buffer Concentrate.

  • To the PCR tube, add 100 l of DNA Binding Buffer.
  • Load the entire contents onto a DNA micro column.
  • Place the column in a 2 ml wash tube.
  • Spin for 1 min at full speed in a microfuge (>10,000 rpm).
  • Discard the flow-through and reuse the wash tube.
  • Add 100 l of DNA Wash Buffer to the column.
  • Spin at full speed (>10,000 rpm) for 1 min.
  • Discard the flow-through and reuse the wash tube.
  • Add 100 l of DNA Wash Buffer to the column.
  • Spin at full speed (>10,000 rpm) for 2 min.
  • Discard the 2 ml wash tube.
  • Place the column containing the sample into an elution tube.
  • Add 8 l of nuclease-free water (pre-warmed to 60C) to the center of the column.
  • Wait for 2 min and spin at full speed (>10,000 rpm) for 1 min.
  • Re-load the filtrate onto the column.
  • Wait for 2 min and spin at full speed (>10,000 rpm) for 1 min.
  • Use the 8 l ds cDNA sample in Step 5 (discard the column).

5. Transcribe the ds cDNA with T7 RNA polymerase in vitro.
Note: Set up the T7 reaction at room temperature.  If Reaction Buffer 3 contains a white precipitate, incubate the buffer at 37C and allow to cool to room temperature before setting up the reaction.

To a PCR tube at room temperature, add the following:

  • Purified ds cDNA                     6.8 l
  • Reaction Buffer 3                    2 l
  • NTP Mix                                7.2 l
  • DTT                                      2 l
  • T7 Booster (Enzyme Mix 3)        2 l
  • Total Reaction Volume              20 l

Incubate in a PCR machine at 37oC for 30-60 mins.
Use 30 mins when the starting total RNA is 1mg and 60 mins when the starting total RNA is 50ng.
Chill on ice for 5 min. and briefly spin to collect all the liquid.
Spin briefly to consolidate the 20 l sample.

6. Purify the amplified RNA (aRNA).

  • To the PCR reaction tube, add 1 l of DNAse.
  • Incubate at 37C for 15 min.
  • Chill on ice for 5 min.
  • Spin briefly to consolidate the 21 l sample.
  • Add 2 l of Sodium Acetate Solution.
  • Add 50 l 100% ethanol.
  • Add 1 l of Linear Acrylamide.
  • Mix gently by pipeting.
  • Incubate at -20C for 20-30 min to precipitate the aRNA.
  • Spin for 15 min (4C) at full speed in a microfuge (>10,000 rpm).
  • Remove the supernatant carefully and discard.
  • Rinse the pellet gently two times with 200 l of 70% ethanol.
  • Dry the aRNA pellet by vacuum centrifugation (can store indefinitely at -80C).
  • Re-suspend aRNA pellet in 25 l nuclease-free water.

7. Perform cDNA synthesis on aRNA using aminoallyl-labeled dNTPs.

  • Obtain 25 l aRNA sample from Step 6.
  • Transfer 2 l in 100 l of water.
  • Quantify spectrophotometrically by measuring the optical density (OD) at 260.
  • Use 1-2 g of aRNA for labeling using the aminoally labeling kit.
  • Use 1-2 g of Universal Reference mRNA as a control for aminoallyl labeling.

Ordering Information

Product

Description

Catalog ID

Price (US dollars)*

Arrayit MiniAmp mRNA Amplification Kit

Arrayit offers a robust and complete kit for human and other eukaryotic RNA amplification applications, optimized for 50 ng-1 g of total cellular RNA including first strand cDNA synthesis, second strand cDNA synthesis, amplification of ds cDNA, purification of ds cDNA, in vitro transcription with T7 RNA polymerase and amplified RNA purification, 10 reactions per kit.

MAK

$942
Purchase1

Arrayit MiniAmp Bacterial mRNA Amplification Kit

Arrayit offers a robust and complete kit for E. coli and other bacterial and prokaryotic RNA amplification applications, optimized for 50 ng-1 g of total cellular RNA including first strand cDNA synthesis, second strand cDNA synthesis, amplification of ds cDNA, purification of ds cDNA, in vitro transcription with T7 RNA polymerase and amplified RNA purification, 10 reactions per kit.

MAKB

$942
Purchase1

To order Arrayit Products: call 408-744-1331, fax 408-744-1711, email arrayit@arrayit.com or click on the purchase button to proceed directly to the purchase page.

*Add shipping and handling to all orders.

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

Warranty
Arrayit Universal mRNA products are sold for research purposes only. Arrayit products are not for use in human diagnostics or for drug purposes, nor are they to be administered to humans in any way.  Extreme care and exact attention should be practiced in the use of the materials described herein.  Arrayit products are subject to extensive quality control and are guaranteed to perform as described when used properly. This product is stable for at least 1 year from the date of purchase when stored at -20C. Any problems with these products should be reported to Arrayit immediately. Our 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 Arrayit makes no warranty or guarantee under such circumstances.

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