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Plant Total RNA Isolation Kit includes two sets of lysis systems, which can solve the RNA extraction of various simple plant tissues, plant tissues rich in polysaccharides and polyphenols, fruit pulp, fungi, etc.During the extraction process of the kit, there is no need to use toxic reagents such as phenol, chloroform, β-mercaptoethanol, or time-consuming alcohol precipitation, and total RNA can be extracted quickly. Product Composition
Plant Total RNA Isolation Kit Cat. No. | 5 preps 5101005 | 50 preps 5101050 |
Filter Columns Spin Columns Buffer RLT Buffer RLC Buffer WA Buffer WBR RNase-free Water Instructions | 5 sets 5 sets 4 ml 4 ml 1.9 ml 1.5 ml 1.5 ml 1 copy | 50 sets 50 sets 32 ml 32 ml 12 ml 10 ml 2 ml×3 1 copy |
Storage and Expiry Date
If the kit is stored at normal temperature (0~30°C), its performance will remain unchanged for two years; if the product is stored at 2~8°C, the validity period of the product can be extended to more than two years.
Introduction
The kit includes two sets of lysis systems, which can solve the RNA extraction of various simple plant tissues, plant tissues rich in polysaccharides and polyphenols, fruit pulp, fungi, etc. During the extraction process of the kit, there is no need to use toxic reagents such as phenol, chloroform, β-mercaptoethanol, or time-consuming alcohol precipitation, and total RNA can be extracted quickly. After ethanol added to the plant tissue lysate, the mixture is added to the spin column. The RNA is bound to the spin column, and the dissolved proteins and PCR inhibitors are filtered out. After the RNA is washed with two washing buffers and eluted with RNase-free water, it can be used in various molecular biology experiments such as RT-PCR, Northern blot, Dot blot, and mRNA isolation.
Equipment and Reagents to Be Supplied by User
1． Absolute ethanol and 70% ethanol
2． 1.5 ml microcentrifuge tubes (RNase-free)
3． Pipettes and tips (to avoid RNase contamination, it is recommended to use RNase-free pipette tips containing filters)
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． Vortexer
7． Laboratories that do not use RNase
Preparation Before Use
1. If the centrifuge has refrigeration function, please set the temperature to 25°C.
2. 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 "Ethanol added".
3. Since saliva and skin contain RNase, disposable gloves and masks are required during the whole process of RNA extraction.
4. If subsequent experiments require complete removal of DNA, please add a DNase I digestion step. The DNase I On-column Digestion Kit (Cat. No. 8010050) needs to be purchased separately.
Lysis Buffer Guidelines
Sample type | Recommended dosage | Sample example | Recommended lysis buffer | |
Buffer RLT | Buffer RLC | |||
simple plant tissue (young leaves, stems, roots) | 50-100 mg | Wheat, rice, corn, Arabidopsis, tobacco, rape, etc. | √ | |
Polysaccharide and polyphenol plant leaves | Cotton leaves, soybean leaves, pine needles, ginkgo leaves, fig leaves, gardenia leaves, etc. | √ | ||
Plant tissue with high starch content | 20-50 mg | Wheat seeds, corn seeds, red bean seeds, potatoes, sweet potatoes, etc. | √ | |
plant tissue with high fat content | Soybean seeds, sesame seeds, peanut seeds, rapeseed, etc. | √ | √ | |
fruit pulp | 100-200 mg | Watermelon, apple, peach, pear, banana, mango, etc. | √ | |
Fungi | 20-100 mg | Shiitake mushrooms, mushrooms, oyster mushrooms, Neurospora crassa, etc. | √ | |
* If you are not sure about the sample type, Buffer RLC can be used first.
Protocol
1. Determine the sample amount and applicable lysis buffer according to the "Lysis Buffer Guidelines". Use liquid nitrogen to grind the tissue to powder. Then use a 1.5 ml microcentrifuge tube pre-cooled with liquid nitrogen to weigh an appropriate amount of tissue and add 600 µl Buffer RLT or 600 µl Buffer RLC , vortex until all tissues are lysed, and centrifuge at 13000 rpm for 2 minutes .
* When grinding tissue, liquid nitrogen should be added in time to avoid melting of the tissue and prevent endogenous RNase from reactivating and degrading RNA.
* Do not use excess tissue, otherwise it may cause clogging of the filter column and contaminate the purified RNA with genomic DNA.
* The nucleic acid content in fresh sprouts is high, and more genomic DNA may remain. DNase I digestion is recommended.
* Buffer RLT and Buffer RLC are corrosive, please wear protective equipment when operating.
2. Pour all the supernatant from step 1 into a filter column, close the lid, and centrifuge at 13000 rpm for 2 minutes.
* If there are a small amount of sample fragments in the supernatant, they can be removed by the filter column and will not affect subsequent experiments.
* 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 5.
3. 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 precipitation after adding 70% ethanol and mixing, please add the precipitation to the spin column together.
4. Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, add pipette the remaining mixture to the spin column, close the lid and centrifuge at 13000 rpm for 1 minute.
* The filtrate does not need to be completely discarded. If you want to avoid the contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can slap the 2 ml collection tube upside down on a paper towel once.
5. Discard the filtrate in the 2 ml collection tube, 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 13000 rpm for 1 minute.
* Make sure absolute ethanol has been added into Buffer WA.
6. Discard the filtrate in the 2 ml collection tube, 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 13000 rpm for 1 minute.
* Make sure absolute ethanol has been added into Buffer WBR.
7. Discard the filtrate in the 2 ml collection tube, place the spin column back into the 2 ml collection tube, and centrifuge at 14000 rpm for 1 minute.
* If the top speed could not reach 14000 rpm, centrifuge at full speed for 2 minutes.
* Do not omit this step, otherwise, it may cause problems in downstream applications due to the residual ethanol in the eluate.
8. 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 in the center of the membrane of the spin column, close the lid and incubate for 1 minute at room temperature, centrifuge at 13000 rpm for 1 minute.
* If the centrifuge does not have a leak-proof cover, please change the centrifugation conditions to 8000 rpm for 1 minute to prevent the 1.5 ml microcentrifuge tube cover from falling off and damaging the centrifuge.
9. Discard the spin column, the eluted RNA can be used immediately for various molecular biology experiments; or the RNA can be stored below -70°C for later use.
