Набор для экстракции небольших количеств тканевой микроРНК

1. Компоненты набора реагентов

2. Хранилище

Этот набор реагентов следует хранить при комнатной температуре. (15-25℃) in a dry condition and is stable for 12 месяцы.

3. Инструкции по использованию набора реагентов

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, водяная баня (металлическая ванна), вихревой смеситель, безводный этанол, liquid nitrogen, chloroform, стерильная деионизированная вода, and EP tubes.

4. Знакомство с набором реагентов

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 час. The extracted microRNA can be directly used for RT-ПЦР, Нозерн-блоттинг, и т. д.. Весь процесс очистки не требует использования токсичных реагентов типа фенол-хлороформа., making the microRNA purification kit suitable for various other samples.

5. Экспериментальные принципы и процедуры

6. Процесс экстракции

Precautions before starting the experiment:

А. Перед использованием, добавьте указанное количество безводного этанола в Стирать Буфер 1 according to the label on the reagent bottle, and mark a check on the label to indicate the addition of anhydrous ethanol.

Б. Элюирующий буфер представляет собой 0.1х ТЕ решение содержащий минимальное количество ЭДТА. Если ЭДТА повлияет на последующие эксперименты, it is recommended to use sterile deionized water instead of Elution Buffer.

1. Образец обработки:

А. 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.

Б. Cell Tissues: Collect fresh growing cells in a 1.5 ml centrifuge tube, центрифуга в 13,000 об/мин для 1 мин, collect cells, добавлять 500μl of RNA Extraction Buffer II, vortex and shake well.

2. Incubate at56℃ for 1-3 мин. 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 об/мин (20,000×г).

(Примечание: 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. После центрифугирования, transfer the supernatant to a new очистка ДНК column.)

5. Add the obtained supernatant to the DNA cleanup column (примерно 650-700 мкл каждый раз), центрифуга при более чем 8,000 об/мин для 1 мин, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, добавлять 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, центрифуга в 13,000 об/мин для 1 мин.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. Добавлять 600μl of Inhibitor Removal Buffer, центрифуга при более чем 8,000 об/мин для 1 мин, discard the waste, and place the RNA purification column back into the collection tube for the next step.
9. Добавлять 700мкл промывочного буфера 1 to the RNA purification column, центрифуга в 14,000 об/мин (20,000×г) для 2 мин, extend the centrifugation time appropriately to ensure a drier membrane.

(Примечание: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. Поэтому, membrane dryness is crucial. После центрифугирования, ensure the absence of ethanol before elution, discard the waste, and collect the tube.

After washing with Wash Buffer 1, the membrane on the RNA purification column should only have a slight color. После центрифугирования, 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, добавлять 100мкл элюирующего буфера to the membrane, incubate at room temperature for 5 мин (15℃ to 25℃), центрифуга при более чем 8,000 об/мин для 1 мин.

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

11. Repeat the previous step.

Примечание: 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.