Small Amount Tissue microRNA Extraction kit

1. Bestandteile des Reagenzienkits

2. Lagerung

Dieses Reagenzienkit sollte bei Raumtemperatur gelagert werden (15-25℃) in a dry condition and is stable for 12 Monate.

3. Anweisungen zur Verwendung des Reagenzienkits

3.1 Dieses Kit ist für molekularbiologische Forschungszwecke bestimmt und sollte nicht zur Diagnose oder Behandlung von Krankheiten verwendet werden.

3.2 Einige Bestandteile des Kits enthalten Reizstoffe; Es ist ratsam, die erforderlichen Vorsichtsmaßnahmen zu treffen (wie das Tragen von Schutzkleidung und Schutzbrillen).

3.3 Die Verwendung dieses Kits erfordert zusätzliche Ausrüstung wie eine Hochgeschwindigkeitszentrifuge, Wasserbad (Metallbad), Vortexmixer, Wasserfreies Ethanol, flüssiger Stickstoff, Chloroform, steriles entionisiertes Wasser, und EP-Röhren.

4. Einführung in das Reagenzienkit

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 Stunde. The extracted microRNA can be directly used for RT-PCR, Northern blotting, usw. The entire purification process does not require toxic reagents such as phenol-chloroform, making the microRNA purification kit suitable for various other samples.

5. Experimentelle Prinzipien und Verfahren

6. Extraktionsprozess

Vorsichtsmaßnahmen vor Beginn des Experiments:

A. Vor Gebrauch, add the specified amount of anhydrous ethanol to Waschen Puffer 1 according to the label on the reagent bottle, and mark a check on the label to indicate the addition of anhydrous ethanol.

B. Elutionspuffer ist ein 0.1x TE-Lösung enthält eine minimale Menge EDTA. Ob EDTA einen Einfluss auf nachfolgende Experimente hat, it is recommended to use sterile deionized water instead of Elution Buffer.

1. Probenverarbeitung:

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, Zentrifuge bei 13,000 U/min für 1 Mindest, collect cells, hinzufügen 500μl of RNA Extraction Buffer II, vortex and shake well.

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

3. Vortex for 30 sec.

4. Zentrifugieren Sie das Lysat 10 min at 14,000 U/min (20,000×g).

(Notiz: 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. Nach Zentrifugation, transfer the supernatant to a new DNA-Reinigung column.)

5. Add the obtained supernatant to the DNA cleanup column (jeweils etwa 650–700 μl), Zentrifuge bei über 8,000 U/min für 1 Mindest, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, hinzufügen 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, Zentrifuge bei 13,000 U/min für 1 Mindest.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. Hinzufügen 600μl of Inhibitor Removal Buffer, Zentrifuge bei über 8,000 U/min für 1 Mindest, Entsorgen Sie den Abfall, and place the RNA purification column back into the collection tube for the next step.
9. Hinzufügen 700μl Waschpuffer 1 to the RNA purification column, Zentrifuge bei 14,000 U/min (20,000×g) für 2 Mindest, extend the centrifugation time appropriately to ensure a drier membrane.

(Notiz: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. daher, membrane dryness is crucial. Nach Zentrifugation, ensure the absence of ethanol before elution, Entsorgen Sie den Abfall, and collect the tube.

After washing with Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. Nach Zentrifugation, 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, hinzufügen 100μl Elutionspuffer to the membrane, incubate at room temperature for 5 Mindest (15℃ to 25℃), Zentrifuge bei über 8,000 U/min für 1 Mindest.

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

11. Repeat the previous step.

Notiz: 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.