Small Amount Tissue microRNA Extraction kit

1. Komponente kompleta reagensa

Tehnički podaci	50T	100T
Mačka. Ne.	SN0333	SN0334
RNA Extraction Columns (set)	50 (set)	100 (set)
DNA Clean-Up Columns (set)	50 (set)	100 (set)
RNA Extraction Buffer II	30ml	2×30 ml
Inhibitor Removal Buffer	30ml	2×30 ml
Pufer za pranje 1	15 ml	2×15 ml
Elucijski pufer	20 ml	20 ml
Priručnik za upute	1	1

 

2. Skladištenje

Ovaj se komplet reagensa treba čuvati na sobnoj temperaturi (15-25℃) in a dry condition and is stable for 12 mjeseca.

3. Upute za korištenje kompleta reagensa

3.1 This kit is intended for molecular biology research purposes and should not be used for disease diagnosis or treatment.

3.2 Some components in the kit contain irritants; it is advisable to take necessary precautions (such as wearing protective clothing and goggles).

3.3 The use of this kit requires additional equipment such as a high-speed centrifuge, vodena kupelji (metalna kupka), vrtlog miksera, bezvodni etanol, liquid nitrogen, chloroform, sterilna deionizirana voda, and EP tubes.

4. Uvod u komplet reagensa

This microRNA purification kit provides a fast and efficient method for purifying microRNA from various tissues, suitable for most species. Tissues exceeding 100 mg can be processed using this RNA purification kit. The kit employs special DNA cleanup column technology to remove genomic DNA and large RNA fragments during the experimental process. In general, additional digestion on DNA columns is not required, preventing microRNA degradation.

The RNA rapid purification kit can extract plant microRNA within 1 sat. The extracted microRNA can be directly used for RT-PCR, Northern blotting, itd. Cijeli postupak pročišćavanja ne zahtijeva toksične reagense kao što je fenol-kloroform, making the microRNA purification kit suitable for various other samples.

5. Eksperimentalni principi i postupci

6. Ekstrakcijski postupak

Precautions before starting the experiment:

A. Prije upotrebe, Dodajte navedenu količinu bezvodnog etanola u Pranje Buffer 1 according to the label on the reagent bottle, and mark a check on the label to indicate the addition of anhydrous ethanol.

B. Buffer za eluciju je a 0.1X TE otopina containing a minimal amount of EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water instead of Elution Buffer.

1. Sample Processing:

A. Plant or Animal Tissues: Collect fresh tissues whenever possible. For plant and animal tissues, grind with liquid nitrogen, then quickly add 500μl of RNA Extraction Buffer II. Invert and mix thoroughly, avoiding tissue clumps in the lysate to prevent RNA degradation.

B. Cell Tissues: Collect fresh growing cells in a 1.5 ml centrifuge tube, centrifuga na 13,000 RPM za 1 min, collect cells, dodati 500μl of RNA Extraction Buffer II, vortex and shake well.

2. Incubate at56℃ za 1-3 min. If the sample has a high polysaccharide content, it is advisable to skip this step.

3. Vortex for 30 sec.

4. Centrifuge the lysate for 10 min at 14,000 rpm (20,000×g).

(Bilješka: Polysaccharides from plant materials may result in sticky substances at this step, which can cut DNA in subsequent steps. Remove these substances during this step. After centrifugation, transfer the supernatant to a new DNA purification column.)

5. Add the obtained supernatant to the DNA cleanup column (approximately 650-700μl each time), centrifuge at over 8,000 RPM za 1 min, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, dodati 0.5 times the volume of absolute ethanol. If there is a small amount of precipitation, it does not affect subsequent experiments. Add the liquid to the RNA purification column, centrifuga na 13,000 RPM za 1 min.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. Dodati 600μl of Inhibitor Removal Buffer, centrifuge at over 8,000 RPM za 1 min, odbaciti otpad, and place the RNA purification column back into the collection tube for the next step.
9. Dodati 700μl pufera za pranje 1 to the RNA purification column, centrifuga na 14,000 rpm (20,000×g) za 2 min, extend the centrifugation time appropriately to ensure a drier membrane.

(Bilješka: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. Stoga, membrane dryness is crucial. After centrifugation, ensure the absence of ethanol before elution, odbaciti otpad, and collect the tube.

After washing with Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. After centrifugation, carefully remove the RNA purification column, ensuring it does not touch the collection tube to avoid ethanol interference.)

10. Place the RNA purification column into a new centrifuge tube, dodati 100μl pufera za eluciju to the membrane, incubate at room temperature for 5 min (15℃ to 25℃), centrifuge at over 8,000 RPM za 1 min.

(Bilješka: Eluting RNA with 50μl of Elution Buffer can increase RNA concentration but decrease total RNA yield.)

11. Repeat the previous step.

Bilješka: A new centrifuge tube can be used to collect the RNA eluted the second time, or the original collection tube can be used to continue collecting RNA.