E.Z.N.A.™Mollusc／ArthropodRNAProtocol

实验概要

The E.Z.N.A.^® Mollusc RNA Kit is designed for efficient recovery of total RNA greater than 200 nt from molluscs, arthropods, roundworms, flatworms, and other invertebrate tissue samples rich in mucopolysaccharides. Samples are homogenized and lysed in RNA-Solv Reagent and extracted with chloroform to remove mucopolysaccharides and denature proteins. Following a rapid alcohol precipitation step, binding conditions are adjusted and RNA further purified using HiBind RNA spin columns. In this way salts, proteins and other contaminants are removed to yield high quality total RNA suitable for downstream applications such as reverse transcription, poly (A)  mRNA selection, and hybridization techniques.

主要试剂

1. Absolute (96%-100%) ethanol

2. Isopropyl alcohol (isopropanol)

3. Liquid nitrogen for freezing/disrupting samples

主要设备

1. Microcentrifuge capable of 12,000 x g

2. Nuclease-free microfuge tubes

3. Preheat an aliquot (100 ul per sample) of DEPC-treated water at 65°C

实验步骤

NOTE: Use extreme caution when handling liquid nitrogen. Use only fresh tissue to ensure RNA integrity. Samples preserved in formalin usually yield degraded RNA but may still yield adequate results for RT-PCR of target regions <500 nt.

Arthropods:

1. Pulverize no more than 50 mg of tissue in liquid nitrogen with mortar and pestle and place the powder in a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (OBI Cat# SSI-1014-39 & SSI-1015-39).Proceed to step 2 below.

Molluscs (and other soft tissued invertebrates):

1. Grind no more than 50 mg tissue in liquid nitrogen with mortar and pestle and place the powder in a clean 1.5 ml microcentrifuge tube. If ceramic mortar and pestle are not available, homogenize the sample in the microfuge tube using a disposable microtube pestle (OBI Cat# SSI-1014-39 & SSI-1015-39). Proceed to step 2 below.

Amount of starting material depends on sample and can be increased if acceptable results are obtained with the suggested 50 mg tissue. For easy to process specimens, the procedure may be scaled up and the volumes of all buffers used increased in proportion. In any event, use no more than 100 mg tissue per HiBind^® spin-column as RNA binding capacity (100 ug) may be exceeded. Meanwhile, difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis.

2. Add 1 ml of RNA-Solv Reagent and vortex vigorously to make sure that all clumps are dispersed. RNA cannot be effectively extracted from clumped tissue. Incubate at room temperature for 2-3 minutes.

3. Add 0.2 mL of chloroform per 1 mL of RNA-Solv Reagent. Cap sample tubes securely and shake vigorously for 15 seconds. Incubate at room temperature for 3 minutes.

4. Centrifuge at 12,000 x g for 15 minutes at 4°C. The mixture separates into a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA remains entirely in the aqueous phase.

5. Transfer no more than 80% of the aqueous phase to a fresh tube. Add one volume of isopropanol and mix to precipitate RNA. Incubate at room tempature for 10 min and centrifuge 12,000 x g for 3 min at 4°C. Carefully discard as much supernatant as possible without disturbing RNA pellet. Invert the microfuge tube on a paper towel for 1 min to allow residual liquid to drain; it is not necessary to dry the pellet.

6. Add 100 ul of sterile DEPC-treated water and vortex to resuspend the pellet. A brief incubation at 65oC may be necessary to effectively dissolve the RNA. Note: All of the centrifugation step below should be carried out at room temperature.

7. Adjust binding condition: Add 350ul Buffer RB/2-mercaptoethanol followed by 250 ul absolute ethanol and vortex to mix. If RB buffer is used in step to dissolve the RNA pellet, add 350ul of 70% ethanol. Vortex to mix.

Note: RB buffer recommended at this step to avoid RNA degradation. Add 20 ul 2-mercaptoethanol per 1 ml of Buffer RB before use. This mixture can be made and stored at room temperature for 1 week.

8. Apply entire mixture, including any precipitation that may have formed, to an HiBind^® RNA column assembled in a 2 ml collecting tube (supplied). Centrifuge .10,000 x g for 30 sec at room temperature. Discard flow-through liquid and reuse collecting tube in next step.

9. Add 500 ul RNA Wash Buffer I and centrifuge at 10,000 x g for 30 sec. Discard both flow-through liquid and collecting tube.

Optional on-membrane DNase I digestion: This is the starting point to perform DNase I digestion. See Page 7 for detailed protocol.

10. Place column in a clean 2ml collection tube (supplied), and add 400 ul Wash Buffer II diluted with ethanol. Centrifuge at 10,000 x g for 30 sec at room temperature and discard flow-through. Reuse the collection tube in step.

Note: Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.

11. Wash column with a second 400 ul of Wash Buffer II as in step 10. Centrifuge and discard flow-through. Then with the collection tube empty, centrifuge the spin cartridge for 2 min at full speed to completely dry the HiBind™ matrix.

12. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 30-50 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution into the same tube may be necessary if the expected yield of RNA >50 ug. To maintain a high RNA concentration, use the first eluate for the second elution.

Note: RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution.