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No need for phenol-chloroform extraction and isopropanol precipitation steps. suitable for isolation of purified total RNA from ≤30 mg tissue/≤1×107 cultured cells. The tissue/cultured cells are lysed in the lysate and released RNA, the RNA is bound to the spin column after adding ethanol, and the dissolved protein and PCR inhibitor are filtered to remove it.complete in 10 mins Filter column + R NA column Product Composition
Animal Tissue/Cultured Cells Total RNA Kit Cat. No. | 5 preps 5001005 | 50 preps 5001050 |
Filter Columns（with 2 ml collection tubes） Spin Columns（with 2 ml collection tubes） β-Mercaptoethanol Buffer RLT Buffer WA (concentrate) Buffer WBR (concentrate) RNase-free Water Instructions | 5 sets 5 sets 50 μl 4 ml 1.9 ml 1.5 ml 1.5 ml 1 copy | 50 sets 50 sets 500 μl 32 ml 12 ml 10 ml 2 ml×3 1 copy |
Product Storage and Expiry Date
If the kit is stored at room temperature (0~30℃), it can maintain no significant change in performance within three years; if the product is stored at 2~8℃, the validity period of the product can be extended to more than three years.
Product Description
This product does not involve the use of phenolic chloroform and is suitable for isolation of purified total RNA from ≤30 mg tissue/≤1×107 cultured cells. The tissue/cultured cells are lysed in the lysate and released RNA, the RNA is bound to the spin column after adding ethanol, and the dissolved protein and PCR inhibitor are filtered to remove it. After RNA is washed by two washing solutions and eluted with RNase-free Water, it can be used in RT-PCR, Northern blot, Dot blot, mRNA isolation and other molecular biology experiments.
Equipment and Reagents to Be Supplied by User
1. Absolute ethanol and 70% ethanol.
2. RNase-free 1.5 ml microcentrifuge tubes
3. Pipettes and pipette tips (RNase-free pipette tips must be selected, DNase-free & RNase-free tips with filter element recommended)
4. Protective equipment such as disposable latex gloves and paper towels
5. Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)
6. Vortex oscillator
7. Laboratory without RNase use
Prepare Before Use
1. If the centrifuge has refrigeration function, please set the temperature to 25℃.
2. Add 10 μl of β-mercaptoethanol to every 1 ml Buffer RLT and mix well. The use of Buffer RLT with β-mercaptoethanol within one month does not affect the experimental results.
3. Add absolute ethanol to Buffer WA and Buffer WBR according to the instructions on the label of the reagent bottle, and tick the box on the label to mark "Alcohol added".
4. Since saliva and skin contain RNase, disposable gloves and masks are required during the whole process of RNA extraction.
Animal Tissue Protocol
1. Weight 200~400 mg of chopped animal tissue to the mortar, grind the sample into powder quickly in liquid nitrogen. Then weigh 20~30 mg powder into a 1.5 ml microcentrifuge tube precooled in liquid nitrogen.
* When grinding tissues, liquid nitrogen should be added in time to avoid tissue melting, to avoid endogenous RNase recovery and degradation of RNA.
* Do not use tissue larger than 30 mg, as this may cause clogging of the filter column and contamination of the purified RNA with genomic DNA.
2. Add 600 μl Buffer RLT which β-mercaptoethanol has been added, vortex and shake until all tissues are dissolved, the solution is translucent, and centrifuge at 13,000 rpm for 2 min.
* Buffer RLT is corrosive, please wear protective equipment to operate.
3. Transfer the supernatant into a filter column, close the lid and centrifuge at 13,000 rpm for 1 min.
* Do not omit this step, as this may cause the spin column to be blocked in subsequent steps.
* If all the lysate cannot be filtered through the filter column, it means that the nucleic acid content in the tissue is too high. At this time, 300 µl filtrate should be transferred to a clean 1.5 ml microcentrifuge tube, and add 300 µl 70% ethanol to the 1.5 ml microcentrifuge tube and mix well by pipetting 6 - 8 times, and then add the entire mixture to a spin column, centrifuge at 13000 rpm for 1 minute. Discard the filter column and remaining filtrate, and continue with step 6.
4. Keep the collection tube and discard the filter column, add 600 μl 70% ethanol to the filtrate and mix well by pipetting 6 - 8 times, pipette 600 µl mixture to a spin column, close the lid and centrifuge at 13000 rpm for 1 minute.
* If there is a precipitate after mixing with 70% ethanol, please add the precipitate together to the spin column.
5. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Transfer the remaining mixture into the spin column, and centrifuge at 13,000 rpm for 1 min.
* The filtrate does not need to be completely discarded, if you want to avoid contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can pat the 2 ml collection tube upside down once on a paper towel.
6. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Add 500 μl Buffer WA to the spin column, close the lid and centrifuge at 13,000 rpm for 1 min.
* Confirm that absolute ethanol has been added to Buffer WA.
7. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Add 600 μl Buffer WBR to the spin column, close the lid and centrifuge at 13,000 rpm for 1 min.
* Confirm that absolute ethanol has been added to Buffer WBR.
8. Discard the filtrate. Place the spin column back into the 2 ml collection tube and centrifuge at 14,000 rpm for 1 min.
* If the centrifuge speed does not reach 14000 rpm, centrifuge at the highest speed for 2 minutes.
* Do not omit this step, otherwise the subsequent RT-PCR effect may be affected by the mixing of ethanol in the purified nucleic acid.
9. Discard the 2 ml collection tube, place the spin column in a clean RNase-free 1.5 ml microcentrifuge tube, add 50~100 μl RNase-free water to the center of the column, close the lid, incubate for 1 minute at room temperature, and centrifuge at 13000 rpm for 1 min.
* If the centrifuge does not have a leak-proof lid, change the centrifugation conditions to 8000 rpm for 1 min to avoid damaging the centrifuge by detaching the tube lid of the 1.5 ml microcentrifuge tube.
10. Discard the spin column. Eluted RNA can be used for a variety of molecular biology experiments or store at -70℃ for later use.
* Even if DNA bands cannot be observed by electrophoresis, purified RNA should not be considered free of genomic DNA contamination, if you need to completely remove DNA, please digest the residual DNA with DNase I.
Culture Cells Protocol
1. Collect ≤1×107 cultured cells in a 1.5 ml microcentrifuge tube and flick the tube to disperse the cells.
* Cell collection method:
a. Cells cultured in suspension: Centrifuge at 300× g for 5 min to collect approximately 1×107 cultured cells and discard the supernatant.
b. Adherent-walled cultured cells: discard the culture supernatant, digest, and suspend the cells with pancrepsin, centrifuge at 300 × g for 5 min to collect about 1×107 cultured cells, discard the pancreatic enzyme supernatant.
c. Cells cultured in a single well in a cell culture plate (if the number of cells in a single well ≤ 1×105, please use the Micro Cells Total RNA Kit: Cat. No.: 5004050): Discard the culture supernatant, directly add 600 μl of Buffer RLT that has been added to β-mercaptoethanol and use the tip to inhalate the cells back and forth several times to lyze the cells, directly into step 3.
2. Add 600 μl Buffer RLT which β-mercaptoethanol has been added and vortex until the cells are completely lysed and the solution is transparent.
* Do not use too many cells, as this may clog the filter column and lead to serious DNA contamination of the final purified RNA genome.
3. Transfer all the cell lysates to a filter column, close the lid, and centrifuge at 13,000 rpm for 2 min.
* Do not omit this step, as this may cause the spin column to be blocked in subsequent steps.
* If there is insufficient liquid filtration at this step, too many cells are being used. In step 4, an equal volume of 70% ethanol with the filtrate can be added to continue the operation, and the other steps remain unchanged.
4. Keep the collection tube and discard the filter column, add 600 μl 70% ethanol to the filtrate and mix well by pipetting 6 - 8 times, transfer 600 μl of the mixture into a spin column, close the lid, and centrifuge at 13000 rpm for 1 minute.
5. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Transfer the remaining mixture into the spin column, and centrifuge at 13,000 rpm for 1 min.
* The filtrate does not need to be completely discarded, if you want to avoid contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can pat the 2 ml collection tube upside down once on a paper towel.
6. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Add 500 μl Buffer WA to the spin column, close the lid, and centrifuge at 13,000 rpm for 1 min.
* Make sure absolute ethanol has been added into Buffer WA.
7. Discard the filtrate. Place the spin column back into the 2 ml collection tube. Add 600 μl Buffer WBR to the spin column, close the lid, and centrifuge at 13,000 rpm for 1 min.
* Make sure absolute ethanol has been added into Buffer WBR.
8. Discard the filtrate. Place the spin column back into the 2 ml collection tube and centrifuge at 14,000 rpm for 1 min.
* If the centrifuge speed does not reach 14000 rpm, centrifuge at the highest speed for 2 minutes.
* Do not omit this step, otherwise the subsequent RT-PCR effect may be affected by the mixing of ethanol in the purified nucleic acid.
9. Discard the 2 ml collection tube, place the spin column in a clean RNase-free 1.5 ml microcentrifuge tube, add 50~100 μl RNase-free water to the center of the column, close the lid, incubate for 1 minute at room temperature, and centrifuge at 12000 rpm for 30 sec.
* If the centrifuge does not have a leak-proof lid, change the centrifugation conditions to 8000 rpm for 1 minute to avoid the tube cap falling off and damaging the centrifuge.
10. Discard the spin column. Eluted RNA can be used for a variety of molecular biology experiments or store at -70℃ for later use.
* Even if the DNA band cannot be observed by electrophoresis detection, it does not mean that the purified RNA does not contain DNA contamination, if you need to completely remove the DNA, please digest the residual DNA with DNase I.
