Small Amount Tissue microRNA Extraction kit

1. 試劑盒的組成

2. 貯存

該試劑盒應在室溫下保存 (15-25℃) in a dry condition and is stable for 12 月.

3. 試劑盒使用說明

3.1 該試劑盒旨在用於分子生物學研究目的，不應用於疾病診斷或治療.

3.2 套件中的某些成分含有刺激物; 建議採取必要的預防措施 (例如穿著防護衣和護目鏡).

3.3 使用該套件需要額外的設備，例如高速離心機, 水浴 (金屬浴), 渦旋混合器, 無水乙醇, 液態氮, 氯仿, 無菌去離子水, 和EP管.

4. 試劑盒簡介

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 小時. The extracted microRNA can be directly used for RT-聚合酶鍊式反應, 北方印跡, ETC. 整個純化過程不需要苯酚氯仿等有毒試劑, making the microRNA purification kit suitable for various other samples.

5. 實驗原理和程序

6. 提取過程

開始實驗前的注意事項:

A. 使用前, 加入規定量的無水乙醇 洗 緩衝 1 根據試劑瓶上的標籤, and mark a check on the label to indicate the addition of anhydrous ethanol.

乙. 洗脫緩衝液是 0.1x TE 解決方案 含有極少量的 EDTA. If EDTA has an impact on subsequent experiments, it is recommended to use sterile deionized water instead of Elution Buffer.

1. 樣品加工:

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.

乙. Cell Tissues: Collect fresh growing cells in a 1.5 毫升離心管, 離心機在 13,000 轉速為 1 分分鐘, collect cells, 添加 500μl of RNA Extraction Buffer II, vortex and shake well.

2. 孵化於56℃ for 1-3 分分鐘. If the sample has a high polysaccharide content, it is advisable to skip this step.

3. Vortex for 30 秒.

4. 將裂解液離心 10 min at 14,000 轉速 (20,000×g).

(筆記: 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. 離心後, transfer the supernatant to a new DNA純化 column.)

5. Add the obtained supernatant to the DNA cleanup column (每次約650-700μl), 離心機超過 8,000 轉速為 1 分分鐘, collect the filtrate (microRNA is in the filtrate at this point).
6. Estimate the volume of the filtrate accurately, 添加 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, 離心機在 13,000 轉速為 1 分分鐘.
7. Discard the waste and place the RNA purification column in a collection tube for the next step.
8. 添加 600µl 抑制劑去除緩衝液, 離心機超過 8,000 轉速為 1 分分鐘, 丟棄廢棄物, and place the RNA purification column back into the collection tube for the next step.
9. 添加 700µl 洗滌緩衝液 1 至RNA純化柱, 離心機在 14,000 轉速 (20,000×g) 為了 2 分分鐘, extend the centrifugation time appropriately to ensure a drier membrane.

(筆記: Confirm the addition of absolute ethanol to Wash Buffer 1. The presence of ethanol has a severe impact on subsequent experiments. 所以, membrane dryness is crucial. 離心後, ensure the absence of ethanol before elution, 丟棄廢棄物, and collect the tube.

After washing with Wash Buffer 1, RNA純化柱上的薄膜應該只有輕微的顏色. 離心後, 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, 添加 100µl 洗脫緩衝液 to the membrane, 室溫孵育 5 分分鐘 (15℃ to 25℃), 離心機超過 8,000 轉速為 1 分分鐘.

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

11. 重複上一步.

筆記: 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.