General RNA Extraction Kit

This kit provides protocols to isolate total RNA from a wide range of sample types, including animal tissues, plant tissues, bacterial, yeast and mammalian cells, viruses, blood, plasma and serum. The Kit procedure provides instruction on lysing the sample to release RNA and stabilizing RNA, followed by binding RNA onto spin columns and removing protein, DNA and other impurities.

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CAD$150.00

  • 50 preps
  • 200 preps

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This kit provides protocols to isolate total RNA from a wide range of sample types, including animal tissues, plant tissues, bacterial, yeast and mammalian cells, viruses, blood, plasma and serum. The first part of procedure is based on the work by Chomczynski and Sacchi ( 1987), namely, samples are lysed and then homogenized in the presence of guanidinium isothiocyanate, a chaotropic salt capable of protecting the RNA by inactivating endogenous RNases. After homogenization, ethanol is added to the sample. The sample is then processed through a spin column containing a clear silica-based-membrane to which the RNA binds. Impurities and DNA do not bind to the column, and are effectively removed by subsequent washing. The purified total RNA is then eluted in RNase-free water and is suitable for use in a variety of downstream applications.


Kit Contents

Component

Quantity

Solution RL

60 ml

Wash Buffer RPI

18 ml

Wash Buffer RW

12 ml

DEPC-treated water

10 ml

RNase-free spin columns

50 each

RNase-free microcentrifuge Collection Tubes

50 each

Materials to be supplied by the users
Chloroform
Ethanol (96100%)

Applications
The isolated RNA using this procedure can b used in:

Real-time reverse transcriptase PCR (RT-PCR)

Reverse transcriptase end-point PCR
Northern blotting
Nuclease protection assays
RNA amplification for microarray analysis
cDNA library preparation after poly(A)+ selection

Isothermal RNA amplification

RNAseq

Features

- Consistent and high yields

- High-quality purified total RNA for downstream applications


Storage
Store at 2-8, protect from light. Kit contents are stable for up to 12 months, when properly stored.

Notes

¬-Wash Buffer RPI and Wash Buffer RW are supplied as concentrates. Before use for the first time, make sure to add ethanol (96100%, molecular biology grade), as instructed on the bottle to obtain a working solution.

-Always use sterile, disposable, and RNase-free plasticware, including, transfer pipettes, pipet tips and microcentrifuge tubes.

-Wear disposable gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves frequently, particularly as the protocol progresses from crude lysates to more purified material.

-Always use proper microbiological aseptic techniques when working with RNA.

-Recommended volume of solution RL (Step 1).

10 cm2 adherent cells

1 ml

107 suspension cells bacteria, yeast, viruses

1-2 ml

100 µl white cells

2 ml

50-100 mg ordinary tissue

1 ml

50-100 mg special tissue (live, spleen, bone or cartilage)

2 ml

15-100 mg plant tissue

1 ml


Protocol

  1. Sample pre-processing
    Tissues
    Tissues from animals or plants (either fresh or frozen at -70°C until use) can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle. Fresh tissues should be minced into small pieces. Homogenize tissue samples in 1 ml Solution RL per 50100 mg tissue using a tissue homogenizer or rotor-stator.
    Alternatively, pass the lysate at least 5 times through a 20-gauge needle (0.9 mm diameter) fitted to an RNase-free syringe. Homogenization shears genomic DNA, reduces the viscosity of the lysates, and increases the yields.

    Adherent Cells
    Lyse cells directly in a culture dish by adding 1 ml of Solution RL to the dish and passing the cell lysate several times through a pipet tip. Note that the amount of Solution RL required is based on the culture dish area (1 ml per 10 cm2) and not on the number of cells present.

    Suspension Cells
    Harvest cells and pellet cells by centrifugation. Use 1 ml of the Solution RL per 510 × 106 animal, plant, or yeast cells, or per 1 × 107 bacterial cells. Lyse cells by repetitive pipetting up and down. Do not wash cells before addition of Solution RL to avoid any mRNA degradation. Disruption of some yeast and bacterial cells may require a homogenizer.

2. Incubate at 15-30°C for 5 minutes, to lyse the nucleoprotein complex completely.

3. (Optional) centrifuge at 12,000 rpm for 5 min at 4°C, transfer the supernatant to a new RNase-free microcentrifuge tube. This step can eliminate protein, fat, polysaccharide, muscle or plant fibre.

4. Add 200 μl chloroform, mix by vortexing for 15 seconds, incubate at room temperature for 3 minutes.

5. Centrifuge the sample at 12,000 rpm for 10 min at 4°C.
Note: After centrifugation, the mixture separates into a lower, yellow phenolchloroform phase, an interphase, and a colorless upper aqueous phase which contains the RNA. Transfer of the colorless, upper phase containing the RNA to a new RNasefree tube.

6. Add an 0.5 volume of ethanol. Mix well, a visible precipitate may form after adding ethanol. Transfer and load the mixture to a spin column with a collection tube (included in the tube), centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through in the collection tube.

7. Add 500 μl Wash Buffer RPI (check whether ethanol is added or not), centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through.

8. Add 500 μl Wash Buffer RW (check whether ethanol is added or not), incubate at room temperature for 1 minutes. Centrifuge at 12,000 rpm for 30 seconds at 4°C, discard the flow-through. Repeat this step once.

9. Centrifuge the column at 12,000 rpm for 2 min to remove any residual liquid from the column. Air dry the column.

10. Place the spin column in a clean 1.5 ml microcentrifuge tube (not provided), and pipet 30-100 μl RNase-free water directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrifuge for 2 min at 12,000 rpm to elute. The tube contains the purified RNA. Store the DNA at -70.

References

Chomczynski P, Sacchi N (1987) Anal Biochem. Apr;162(1):156-9. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

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