1. INTRODUCTION
The RNA STAT "SINGLE STEP METHOD" Affinity chromatography RNA purification kit has been developed for use with the "single step RNA isolation method" ¹. The RNA STAT kit utilizes a proprietary coated resin that effectively absorbs RNA while allowing contaminants such as guanidinium, phenol, detergents, salts, polysaccharides and glycogen to wash through an easy to use push column. The RNA STAT kit provides columns that quickly and effectively, eliminate contaminants from "single step method" RNA preps. These contaminants can cause high background levels, lead to inaccurate quantitation of RNA, inhibit enzymes, or adversely affect sample activity and consistency². The RNA STAT kit simplifies the "single step RNA isolation method" by eliminating the 15 minute 4^oC precipitation and subsequent 15 minute 12,000g centrifugation steps. The 8 minute centrifugation step following the 75% ethanol wash is also eliminated as is the 15 minute pellet drying step. Additionally, the 10-15 minute step to heat and dissolve the RNA pellet(s) is not required. The RNA STAT kit consistently provides high quality undegraded RNA preps that are free from contaminating guanidinium, phenol, detergents, salts, proteins, polysaccharides, glycogen and other non-RNA contaminants offering:
*Purer RNA
*Reduces "single step RNA isolation method" by one hour
*Direct RNA binding to anion exchange resin
*In situ RNA washing and manipulation
*Greater prep to prep consistency
*No pellets to dissolve. No reprecipitation step
*Enzymatic assay ready RNA in 30 minutes
*Facilitates RNA extraction from gels

2. KIT COMPONENTS                                SIZE                          STORAGE
TL 1001 Solution 1 Washing Reagent               60ml                                   2-8 ^oC
TL 1002 Solution 2 Equilibration Reagent         60 ml                                  2-8 ^oC
TL 1003 Solution 3 Binding Reagent                 30ml                                   2-8 ^oC
TL 1004 Solution 4 Elution Reagent                  60ml                                   2-8 ^oC
TL 1005 Solution 5 DEPC Ultra pure water      60ml                                   2-8 ^oC
TL 1006 Affinity Push Columns                           50                                     2-8 ^oC
TL 1007 Collection Tubes                                   50                                     2-8 ^oC
TL 1008 3cc Syringes                                         50                                     2-8 ^oC

ITEMS REQUIRED BUT NOT SUPPLIED
RNAzol, RNAzol B, RNA STAT-60, or equivalent single step method reagent(s), chloroform (ACS grade) and ethanol (ACS grade).

3. METHOD
Total RNA isolation by RNA STAT "SINGLE STEP METHOD" kit includes the following steps:
1. Homogenization As per "single step method" (50-100 mg tissue, or 5-10 x 10⁶ cells/column)
2. RNA Extraction As per "single step method"
3. Column Washing Add 1ml of washing reagent to column
4. Column Equilibration Add 1ml of equilibration reagent to column
5. RNA Immobilization Add 0.25 vol. column binding reagent to column
6. RNA Wash Wash immobilized RNA with 75% ethanol
7. RNA Elution Elute RNA from column with 200 ul of elution buffer or DEPC water

3.1 COLUMN WASHING
Note: Use one column per 50-100mg tissue or 5-10⁶ cells.
1. Remove syringe plunger from syringe barrel and attach syringe to the column luer lock.
2. Wash each column with 1ml of Solution 1 (washing reagent). Fill the syringe with 1ml of Solution 1 (washing reagent) and eject reagent through column by depressing the syringe plunger. Discard flow-through.

3.2 COLUMN EQUILIBRATION
1. Equilibrate each column with 1ml of Solution 2, (equilibration reagent). Fill the syringe with 1ml of Solution 2 (equilibration reagent) and eject reagent through column by depressing syringe plunger. Discard flow-through.

3.3 RNA IMMOBILIZATION
1. Transfer the aqueous phase containing RNA to a fresh tube and add 0.25 volume of Solution 3 binding reagent and 2 volumes (original aqueous phase) of 100% ethanol. Gently mix with pipet and store at room temperature for 5 minutes.
2. Remove syringe plunger from syringe barrel and attach syringe barrel to the column luer lock.
3. Add RNA sample to syringe. Each RNA sample slowly (1 drop/second) through column by depressing syringe plunger. Discard flow-through.Note: The RNA sample flow-through can be saved to later verify column binding efficiency.
The RNA is now bound and immobilized on the column.

3.4 RNA WASH
1. Remove syringe plunger from syringe barrel and attach syringe barrel to the column luer lock.
2. Wash column bound RNA with 1ml of 75% ethanol. Eject ethanol through column by depressing syringe plunger. Discard flow-through.
3. Dry the immobilized RNA briefly by air drying for 5-10 minutes or by blowing air through the column with a syringe.

3.5 RNA ELUTION
1. Transfer column to RNase free 1.5ml collection tube (provided).
Elute column bound RNA with 200ul of Solution 4 (elution reagent). (See Section 4.3) Push elution reagent slowly (1 drop/second) through the column using the syringe plunger and collect RNA. Repeat a second time using a fresh RNase free 1.5ml microfuge tube.
Measure 260/280 ratios of each 200ul fraction and combine as necessary to maximize yield.
4. NOTES AND COMMENTS
1. The column bound and immobilized RNA can be eluted with most any appropriate RNase free buffer required for subsequent procedures.
2. To concentrate the RNA, pass original (100ul-200ul) fraction through column 3 times.
3. Solution 5 (DEPC water) may be substituted for Solution 4 (elution buffer).
4. The mini columns can achieve RNA recovery rates of up to 99%.
5. Hands and dust may be the major source of the RNase contamination. Use gloves and keep tubes closed. The use of sterile, disposable polypropylene tubes is recommended throughout the procedure.

5. REFERENCES
1. Chomcyznski P. and Sacchi N. (1987) Single-Step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159.2. Puissasnt, C. and L. Houdebine, 1991. An improvement of the Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol Chloroform Extraction. Biotechniques. 8:148-149.3. Sambrook J., Fritsch E.F. and Maniatis T. (1989) Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.4. Kedzierski, W. and John Porter (1991). A Novel Nonenzymatic Procedure for Removing DNA Template for RNA Transcription Mixtures. Biotechniques. 10:210-214.

RNA-STAT, RNA STAT-60, and RNA STAT-30 are trademarks of Tel-Test "B", Inc.

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