I am trying to isolate RNA from Trizol and triton x but results are not coming, so help me out. Thanks
Sir the method uses the qiagen RNeasy kit but you can use the pretreatment TE buffer mentioned in supplementary information below.
Plz. refer the link for full article [RNA-Seq-based transcriptome analysis of methicillin-resistant Staphylococcus aureus biofilm inhibition by ursolic acid and resveratrol]
http://www.nature.com/srep/2014/140627/srep05467/full/srep05467.html
Total RNA isolation
All samples used for the RNA sequencing were prepared in the 24-well flat bottomed polystyrene plates. After the biofilms were rinsed, the compound-free and treated biofilm cells were scraped with pipettor and placed in the RNAprotect Bacteria Reagent (QIAGEN GmbH, Germany). The sessile cell suspension was then transferred to a microcentrifuge tube and incubated for 5 min at room temperature to stabilize the mRNA. Next, the cell suspensions were centrifuged at 8,000 × g for 5 min to pellet the cells, and the supernatant was decanted. Total RNA was purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's recommended protocol with some modifications (Supplementary materials* online). Each total RNA sample was suspended in 30 μL of RNA storage solution and the quality of total RNA obtained was determined using Agilent 2100 bioanalyzer.
*Supplementry file material :Total RNA isolation
Procedures of using RNeasy Mini Kit to purify MRSA total RNA:
1. Prepare TE buffer (30 mM Tris·Cl, 1 mM EDTA, pH 8.0) containing 20 mg/mL lysozyme and 12.5 μg/mL lysostaphin.
2. Add 200μL Proteinase K and 200 μL TE buffer (step 1) to the pellet treated by RNA Protect solution.
3. Mix by vortexing for 10 s. Incubate in the bath at 37°C for 1 h. During incubation, vortex for 10 s at least every 5-10 min.
4. Add 700 μL Buffer RLT (Ensure that β-meracaptoethanol is added to Buffer RLT before use). Vortex vigorously for 5–10 s. Centrifuge for 2 min at maximum speed. Transfer supernatant into a new tube.
5. Add 500 μL ethanol (100%). Mix well by pipetting. Do not centrifuge.
6. Transfer up to 700 μL lysate, including any precipitate that may have formed, to an RNeasy Mini spin column placed in a 2 ml collection tube. Close the lid gently, and centrifuge for 15 s at 12,000 ×g. Discard the flow-through. Reuse the collection tube in step 7. If the lysate exceeds 700 μL, centrifuge successive aliquots through the spin column. Discard the flow-through after each centrifugation.
7. Add 350 μL Buffer RW1 to the RNeasy Mini spin column. Close the lid gently, and centrifuge for 15 s at 12,000 ×g to wash the spin column membrane. Discard the flow-through and collection tube.
8. Add 80μL DNase I solution to the RNeasy Mini spin column. Incubate at room temperature (15–25°C) for 15 min.
9. Add 350 μL Buffer RW1 to the RNeasy Mini spin column. Incubate at room temperature (15–25°C) for 5 min. Discard the flow-through and collection tube.
10. Place the RNeasy Mini spin column in a new 2 ml collection tube. Add 500 μL Buffer RPE (Ensure that ethanol is added to RPE buffer before use) to the RNeasy Mini spin column. Close the lid gently, and centrifuge for 15 s at 12,000 ×g to wash the spin column membrane. Discard the flow-through. Reuse the collection tube in step 11.
11. Add 500 μL Buffer RPE to the RNeasy Mini spin column. Close the lid gently, and centrifuge for 15 s at 12,000 ×g to wash the spin column membrane. Discard the flow-through. Reuse the collection tube.
12. Centrifuge for 2 min at maximum speed, ensures that no ethanol is carried.
13. Place the RNeasy Mini spin column in a new 1.5 mL collection tube. Open the lid and incubate at room temperature (15–25°C) for 2 min. Add 30 μL preheated RNA storage solution directly to the spin column membrane. Close the lid gently and incubate at room temperature (15–25°C) for 2 min. Centrifuge for 1 min at 12,000 ×g to elute the RNA.
14. Suction out the elutate and then add to the RNeasy Mini spin column. Close the lid gently and incubate at room temperature (15–25°C) for 2 min. Centrifuge for 1 min at 12,000 ×g. The collection solution is the total RNA.
hello aseem thanks for reply, but aseem they are again using kit, we cant afford kit that is why working on some manual methods.
Sir
RNeasy kits use guanidine thiocynide or guanidine hydrochloride based reagents in their buffers. trizol also contains the same reagents. you may use their TE buffer (supplementry material) for pretreatment of cells and then proceed with trizol method. in cases in which trizol doesn't work , please use phenol-SDS method.
Dear aseem they use columns , i dont have columns too, but still i will try this method. Thanks
Many current methods isolation of RNA from Gram-positive bacteria are based on enzymatic lysis, or required disruption procedures by mechanical disruption of cell wall by beads or sonication or by combination of them. These methods have been effected and having the model does not work well with bacterial producing biofilm. They are labor-intensive due to difficult handling and the need to perform additional precipitation steps which affect the total RNA isolation and result in low-integrity RNA.
I try many protocols to isolate RNA from MRSA. the best method is lysis the cell wall using Lysostaphin 200 um/ ml, and continue with Acid phenol chloroform ( 1: 1).
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