產品介紹

E.Z.N.A.? MicroElute? Total RNA Kit provides a rapid and easy method for the isolation of up to 50 μg of total RNA from small amounts of cultured eukaryotic cells, tissues such as laser dissected samples (LDS) or fine-needle aspirates (FNA). Normally, up to 5 x 105 eukaryotic cells or 5 mg tissue can be used in a single experiment depending on the type of tissue used. This kit allows the processing of single or multiple samples in less than 30 minutes. There is no need for phenol/chloroform extractions, and time-consuming steps such as CsCl gradient ultracentrifugation, and precipitation with isopropanol or LiCl. Purified RNA can be eluted with 10-15 μL nuclease-free water. Purified RNA is ready for most downstream applications such as RT-PCR, Northern blotting, poly A+ purification, nuclease protection and in vitro translation.

參數

組分

流程

RNA Purification from Laser Dissected Samples

1. Add 300 μL TRK Lysis Buffer mixed with 2-mercaptoethanol to a 1.5 mL or 2 mL microcentrifuge tube (not provided).
   Optional: If using <5,000 cells, add 4 μL (20 ng) Carrier RNA Stock Solution before homogenization.

2. Transfer homogenized sample to the microcentrifuge tube containing TRK Lysis Buffer.

3. Adjust the volume to 350 μL with TRK Lysis Buffer. Vortex for 30 seconds to mix thoroughly.

4. Add 1 volume 70% ethanol. Vortex to mix thoroughly. Do not centrifuge.
   Note: A precipitate may form at this point. This will not interfere with the RNA purification.

5. Insert a MicroElute? LE RNA Column into a 2 mL Collection Tube and follow the column equilibration steps listed below:
   Protocol for Column Equilibration:

       1. Add 100 μL 3M NaOH to the MicroElute? LE RNA Column.

       2. Centrifuge at 10,000g for 30 seconds.

       3. Add 500 μL sterile deionized water to the MicroElute? LE RNA Column.

      4. Centrifuge at 10,000g for 30 seconds.

      5. Discard the filtrate and reuse the collection tube.

6. Transfer the entire lysate from Step 4, including any precipitates that may have formed, to the MicroElute? LE RNA Column.

7. Centrifuge at maximum speed (≥13,000g) for 15 seconds at room temperature. Discard the filtrate and the collection tube.
   Optional: This is the starting point of the optional on-membrane DNase I Digestion Protocol. Since the HiBind? matrix of the MicroElute? LE RNA Column eliminates most DNA, DNase I digestion is not necessary for most downstream applications. However, certain sensitive RNA applications may require further DNA removal. If an additional DNA removal step is required, please continue to the DNase I Digestion Protocol found on page 22 of the product manual. (See DNase I Digestion Set, (E1091) for more information). If DNase I digestion is not required, proceed to Step 8.

8. Transfer the MicroElute? LE RNA Column to a new 2 mL Collection Tube.

9. Add 500 μL RWF Wash Buffer. Centrifuge at maximum speed for 30 seconds. Discard the filtrate and reuse the collection tube.

10. Add 500 μL RNA Wash Buffer II diluted with 100% ethanol (see the bottle for instructions). Centrifuge at maximum speed for 30 seconds. Discard the filtrate and reuse the collection tube.

11. Repeat Step 10 for a second RNA Wash Buffer II wash step.

12. Centrifuge at maximum speed for 2 minutes to completely dry the MicroElute? LE RNA Column.
    Note: It is important to dry the MicroElute? LE RNA Column matrix before elution. Residual ethanol may interfere with downstream applications.

13. Transfer the MicroElute? LE RNA Column to a clean 1.5 mL microcentrifuge tube.

14. Add 15-20 μL Nuclease-free Water directly onto the MicroElute? LE RNA Column matrix. Centrifuge at maximum speed for 1 minute.

15. Store eluted RNA at -70°C.

RNA Purification from Animal Tissue or Cell Cultures
Optional: Prepare Carrier RNA Stock Solution according to the table on the reverse page and store in aliquots at -70°C.

1. Determine the amount of starting material and homogenize the samples. Do not use more than 5 mg tissue or 5 x 10? cells. If using <5,000 cells, add 4 μL (20 ng) Carrier RNA Stock Solution before homogenization.

2. Add 350 μL TRK Lysis Buffer to a 1.5 mL or 2 mL microcentrifuge tube (not provided).
   Note: Add 20 μL 2-mercaptoethanol per 1 mL TRK Lysis Buffer before use.

3. Transfer homogenized sample to the microcentrifuge tube containing TRK Lysis Buffer. Vortex for 30 seconds to mix thoroughly.

4. Centrifuge at maximum speed (≥13,000g) for 2 minutes.

5. Transfer the cleared supernatant to a new microcentrifuge tube.

6. Add 1 volume 70% ethanol. Vortex to mix thoroughly. Do not centrifuge. A precipitate may form at this point. This will not interfere with the RNA purification.

7. Proceed to Step 5 of the RNA PURIFICATION FROM LASER DISSECTED SAMPLES protocol on the reverse page.