INSTRUCTION MANUAL

ZR Whole-Blood Total RNA Kit

Catalog Nos. (R1020 & R1021)

Contents
I. General Information 1 - 3 Description Highlights Specifications Product Contents Ordering Information II. Protocol.. 3 - 5 Notes Troubleshooting III. List of Products....6

For Laboratory Use Only

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I. General Information
Description
Zymo Researchs ZR Whole-Blood Total RNA Kit provides a hassle-free method for the rapid isolation of total RNA from whole-blood in as little as 10 minutes. This simple procedure is based on the use of a unique single-buffer system combined with Fast-Spin column technology. The procedure is easy: just add the buffer to a blood sample and the extracted RNA is then isolated in RNase-free water using a Zymo-Spin IIIC Column. Optionally, the RNA can be further purified and concentrated into 6-10 l of RNase-free water using a Zymo-Spin IC Column. RNA can be isolated immediately from fresh samples or at a later time from blood stored in ZR RNA Buffer. This product is designed to purify and concentrate RNA from blood for subsequent RNA-based methods including RT-PCR, hybridization, etc.

Highlights
Convenient method for purifying total RNA from whole-blood samples. Compatible with commonly used anticoagulants (i.e., EDTA, heparin, citrate). Allows RNA to be eluted at high concentrations into minimal volumes of RNase-free water in just minutes. Purified RNA is suitable for use in RNA-based procedures including RT- PCR. Omits the use of organic denaturants and proteinases.

Specifications
RNA Purity High quality, purified RNA is eluted into RNase-free water and is suitable for RNA-based manipulations including RT-PCR. RNA Recovery Typically, RNA is eluted into 40 l RNase-free water or (optionally) 6-10 l for a highly concentrated sample. The RNA binding capacity of the Zymo-Spin IC Column is 5 g. RNA Storage Recommended that 1 U/10 l RNase inhibitor be added to the RNA prior to storage at -70oC. Sample Sources Whole-blood (fresh or stored in ZR RNA Buffer). Stability of Product Reagents Integrity of kit components is guaranteed for up to one year from date of purchase. Quality Control Reagents are routinely tested on a lot-to-lot basis to ensure they provide maximal performance and reliability.

Note: Satisfaction of all Zymo Research products is guaranteed. If you should be dissatisfied with this product please call 1-888882-9682.

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Product Contents
ZR Whole-Blood Total RNA Kit Kit Size ZR RNA Buffer RNA Pre-Wash Buffer RNA Wash Buffer* DNase/RNase-Free Water Zymo-Spin IC Columns Zymo-Spin IIIC Columns Collection Tubes RNase-Free Tubes Instruction Manual R1020 (50 Preps.) 50 ml 25 ml 24 ml 6 ml 50 ct. 50 ct. 3x50 ct. 2x50 ct. 1 R1021 (100 Preps.) 100 ml 50 ml 24 ml 2x6 ml 2x50 ct. 2x50 ct. 6x50 ct. 1 Storage Temperature Room Temp. Room Temp. Room Temp. Room Temp. Room Temp. Room Temp. Room Temp. Room Temp. -

* Ethanol must be added prior to use as indicated on RNA Wash Buffer label.

Ordering Information
Product Description ZR Whole-Blood Total RNA Kit ZR Whole-Blood Total RNA Kit For Individual Sale ZR RNA Buffer RNA Pre-Wash Buffer Catalog No. R1020 R1021 Catalog No. R1020-1-50 R1020-1-100 R1020-2-25 R1020-2-50 R1003-3-2.4 R1003-3-6 R1003-3-12 R1003-3-24 R1003-3-48 W1001-1 W1001-4 W1001-6 W1001-10 C1004-50 C1004-250 C1006-50 C1006-250 Kit Size 50 preps. 100 preps. Amount 50 ml 100 ml 25 ml 50 ml 2.4 ml 6 ml 12 ml 24 ml 48 ml 1 ml 4 ml 6 ml 10 ml 50 ct. 250 ct. 50 ct. 250 ct.

RNA Wash Buffer (concentrate)

DNase/RNase-Free Water

Zymo-Spin IC Columns Zymo-Spin IIIC Columns

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For Individual Sale Cont. Collection Tubes RNase-Free Tubes

Catalog No. C1001-50 C1001-500 C1001-1000 C2001-50

Amount 50 ct. 500 ct. 1,000 ct. 50 ct.

For Technical Assistance contact those at Zymo Researchs Technical Department at 1-888-8829682 or E-mail to tech@zymoresearch.com.

II. Protocol
Overview
The ZR Whole-Blood Total RNA Kit is a convenient method for purifying RNA from fresh or stored whole-blood samples in ZR RNA Buffer using Zymo Researchs Fast-Spin column technology. As shown below, RNA is isolated using a Zymo-Spin IIIC Column and then (optionally) concentrated using a Zymo-Spin IC Column. In the latter step, the RNA is washed twice then eluted with a minimal volume (8-10 l) of RNase-free water. The entire procedure typically takes about 1015 minutes.
Note: Immediate processing of blood samples is recommended. Should storage of blood be required prior to processing, see Note 2 on Page 5.

Reagent Preparation
Before starting, add 24 ml 100% ethanol to the 6 ml RNA Wash Buffer concentrate (96 ml 100% ethanol to the 24 ml wash buffer concentrate) to obtain the final RNA Wash Buffer solution. Make sure guidelines are followed to ensure the RNA isolation procedure is performed in an RNase-free environment.
Note: Alternatively, add 26 ml and 104 ml of 95% ethanol to the 6 ml and 24 ml sizes of the wash buffer concentrate, respectively.

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Method
The following protocol is based on a 200 l sample volume of blood. 1. Add 600 l ZR RNA Buffer to 200 l whole-blood sample and mix. 2. Transfer the mixture to a Zymo-spin IIIC Column in a Collection Tube. 3. Centrifuge >10,000 rpm (10,000-12,000 x g) for 30-60 seconds. Discard the Collection Tube containing the flow-through. 4. Add 400 l of RNA Prewash Buffer to the column. Centrifuge at >10,000 rpm for 30-60 seconds. Discard flow-through. 5. Add 400 l of RNA Wash Buffer to the column. Centrifuge at >10,000 rpm for 30-60 seconds. Discard flow-through. 6. Add 50 l of DNase/RNase-Free Water directly to the column matrix. Place column into an RNase-free 1.5 ml tube. Centrifuge briefly to elute the RNA. The RNA is ready for use at this time; however, if the RNA requires concentration, follow the steps below. (optional) 1. Add 400 l of ZR RNA Buffer to the eluted RNA and mix. 2. Transfer the mixture to a Zymo-Spin IC Column in a Collection Tube. 3. Centrifuge at >10,000 rpm for 30-60 seconds. Discard the flow-through. 4. Add 200 l of RNA Wash Buffer to the column. Centrifuge at >10,000 rpm for 30-60 seconds. Discard flow-through. Repeat wash step. 5. Add 8-10 l of DNase/RNase-Free Water directly to the column matrix. Place column into an RNase-free 1.5 ml tube. Centrifuge at >10,000 rpm for 30-60 seconds to elute the RNA. The purified RNA can be used immediately or stored at -70oC.
Note: Water is strongly recommended to elute the RNA. TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0) can also be used for elution if required by your experiment. Waiting for one minute prior to eluting the RNA may increase RNA yield. Also, the yield may be increased by performing a second elution with another 6-8 l of water and pooling it with the first. Note: The amount of blood used can be adjusted depending on the application. Should this be the case, adjust step 1 accordingly and follow the other steps as directed (See Note 1 on Page 5). If the blood sample cant be processed immediately, the blood can be stabilized for processing at a later time (see Note 2 on Page 5).

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Notes
1. RNA Purification Using Different Volumes of Whole-Blood. If RNA needs to be purified from different volumes of blood than that indicated in the standard Protocol (200 l), simply add 3 volumes of ZR RNA Buffer to every volume of whole blood used. (For 150 l blood, add 450 l of ZR RNA Buffer). Adhere to the other steps as indicated in the protocol. One Zymo-Spin IIIC Column can process up to 400 l blood through sequential loading of sample. 2. Delayed Processing of Blood Samples: Immediate processing of blood for RNA purification is highly recommended. However, if the blood cannot be processed immediately, the sample can be stabilized in ZR RNA Buffer for RNA purification at a later time. To do this, simply add 3 volumes of ZR RNA Buffer to every volume of whole-blood. Blood samples mixed with ZR RNA Buffer can be stored at room temperature for 2 days, 0-4 C for up to 5 days, -20 C for up to 4 weeks, and -70 C for over 6 months. After allowing a stabilized sample to reach room temperature, proceed with step 2 in the standard protocol.

Troubleshooting
1. RNA Degradation: Check for RNase contamination of buffers. All buffers and contents supplied by Zymo Research are certified RNase-free. RNases may be introduced during execution of the purification procedure. Exercise all necessary precautions to ensure the procedure is conducted in an RNase-free environment. 2. Problems with RNA in Subsequent Experiments: Make sure to add 3 volumes of ZR RNA Buffer to every volume of whole-blood. Also, make sure spin procedures are complete, as incomplete washing of salts and buffers may adversely affect the outcome of the procedure. 3. DNA Contamination: The combination of buffers and columns provided in this kit are optimized to efficiently isolate and purify RNA and exclude DNA. Additional DNA removal may be required if subsequent applications are affected by trace amounts of DNA. This can be accomplished using the DNAFree RNA kit (Cat. Nos. R1013 & R1014) from Zymo Research.

Note - Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by trained professionals. Some reagents included with this kit are chaotropic and are irritants. Wear protective gloves and eye protection. Follow the safety guidelines and rules enacted by your research institution or facility.

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