Worthington RNA determination detailed introduction

AI Meijie Worthington  RNA background ：

RNA is through  3',5'- Long chain polymers of phosphodiester linked nucleotides . The constituent bases are derivatives of purine and pyrimidine , Sugar is  D- Ribose .RNA  The nucleotide sequence is from  DNA  Genetic information transmitted , As a template for amino acid sequencing in protein synthesis . See  JD Watson  Molecular biology of genes  3  edition （WA Benjamin, Inc., Menlo Park, California 1975） No  11  Chapter  - DNA  On the template  RNA  Transcription and  12  Chapter  - RNA  Involved in protein synthesis . Ribonucleic acid core is composed of limited polynucleotides remaining after complete ribonuclease hydrolysis of yeast sodium nucleic acid .

AI Meijie Worthington  RNA Chemical structure ：

 

AI Meijie Worthington  RNA Determination ：

Method :  By means of  RNase  Used as a substrate in determination to determine as  RNase  Applicability of substrate . Due to different reaction rates and separation from biological sources  RNA  Significant differences in nucleotide patterns , Standardization of ribonuclease activity has been difficult . Crook et al .(1960)  Published the use of synthetic substrates  2', 3'- Determination of cytidine phosphate .Zimmerman  and  Sandeen (1965)  A sensitive determination using cytidine acid is described .

Kalnitsky  People's method .（1959） Used in Worthington . yeast  RNA  stay  pH 5.0  The hydrolysis rate of is determined by measuring the amount of acid soluble oligonucleotides released under specified conditions . Under specified conditions , One unit will cause  A260  stay  37°C  and  pH 5.0  The absorbance increases  1.0.

Reagents ：

0.10 M  Sodium acetate buffer ,pH 5.0： take  5.75 ml  Glacial acetic acid is dissolved in  900 ml  Reagent grade water . use  5 N NaOH  take  pH  Adjust the value to  5.0, And use reagent grade water to make the final volume reach  1000 ml.

contain  0.75%  Uranyl acetate  25%  Perchloric acid ： take  50 ml 70%  Perchloric acid  (HClO 4 )  Dissolve in  90 ml  Reagent grade water . add to  750 mg  Uranyl acetate  (MW 424.15)  And stir to dissolve .

RNA  solution ： take  100 mg Worthington  RNA  (RNA)  Dissolve in  10 ml 0.1 M  In sodium acetate buffer ,pH 5.0  Stir gently to dissolve to prevent denaturation . stay  0.10M  Sodium acetate  pH 5.0  China and Israel  1:50  Dilution record  A260, To determine  mg RNA/ml.mg RNA/ml = A260 x  Dilution  x 0.04

stay  0.10M  Dilute sodium acetate to  2.5 mg/ml RNA,pH 5.0  Used to determine . Before determination, at  37°C  Next incubation  5-10  minute . Prepare a copy to be tested  RNA  solution , And another different batch  RNA  The solution of is used as the control .

RNAse：

Prepare in reagent grade water  1 mg/ml  Stock solution .

Just before the determination , stay  0.10 M  Sodium acetate ,pH 5.0  Further dilute to per ml  2、4  and  6  MCG .

Program ：

determine  % Native RNA： take  10 mg RNA  Dissolve in  10 ml 0.015% NaCl  in . stay  NaCl  Press  1:50  Dilution , take  3 ml  Move the pipette into the cuvette , stay  260 nm  and  320 nm  Readings at . Join in  0.2 ml 5N NaOH（ fresh , Store in plastic ）. stay  260  and  320 nm  Read at . stay  37°C  Let it stand for an hour . stay  260  and  320 nm  Read at . determine  % Native：（ If lower  60%, Please contact your supervisor .）.

A320  It's a check of irrelevant background .

RNase Blank Assay：（ It can be done by  RNase  Detection settings ） For samples  RNA  Set blank , For testing  RNA  Set blank . take  1 ml 0.10M  Sodium acetate buffer  pH 5.0  Move into the centrifuge tube . Balance to  37°C. Add one milliliter  RNA  solution （2.5  mg / ml ）. Let us in  37°C  Set up an hour . Then add 1 ml of uranyl acetate - Perchloric acid solution . Transfer to an ice bath to cool  5  minute . Centrifuge to clarify and dissolve  0.1ml  The clarified supernatant is diluted to  3.0ml. read  A260  And blank . stay  37°C  Next , The blank should not be increased by more than  50%.

RNase Assay： Set up a set of test tubes for the sample , Set up a set of tubes for the control . Including blank with sample tube group and blank with control tube group . Transfer 1 ml of the corresponding enzyme diluent into a centrifuge tube ; learn  1 ml 0.10M  Sodium acetate buffer  pH 5.0  Into the blank tube . Place all tubes in  37°C  Next incubation  58  minute . Every once in a while , take  1 ml RNA  solution  (2.5 mg/ml)  Add to all tubes （ sample 、 Contrast and blank ） in . Incubate each tube accurately  4  minute , Join in  1 ml  Uranyl acetate - Perchloric acid solution terminates the reaction . Transfer to an ice bath to cool  5  minute . Centrifuge to clarify and dissolve  0.1 ml  Dilute the clarified supernatant to  3.0 ml. read  A260  And blank .

Calculation

Company /mg dw = (A260 -  blank ) x 30 x  Dilution  x  Standardization factor

Be careful ： As a substrate  RNA  From natural sources , Therefore, there will be differences between batches . To overcome this , Run the ribonuclease standard and adjust the value to standard . Example ： If the value assigned to the standard is  2500 u/mg, The standard value obtained is  2000 u/mg, Will  1.25  The coefficient of shall be applied to the test value .

Worthington Core enzyme ： Include ： Collagenase , Deoxyribonuclease I, Elastase , Papain , Ribonuclease , Trypsin, etc .