Real-Time PCR experiment process........

Experimental Reagents - **Total RNA Kit**: Omega (Cat. No. R6834) - **First Strand cDNA Synthesis Kit**: GeneCopoeia (Cat. No. C0210A) - **2x AllinOne™ Q-PCR Mix**: GeneCopoeia (Cat. No. D0101A) - **Primer Synthesis**: Invitrogen Experimental Apparatus - **Real-Time PCR Machine**: ABI Experimental Materials The sample consisted of a cell culture solution derived from five human sources, labeled as No. 1 to No. 5. Among these, No. 1 served as the control group, while Nos. 2 to 5 were experimental groups representing different conditions or treatments. Experimental Procedure **1. RNA Extraction** 1) Sample Processing: Add an appropriate volume of the sample to 1 mL of TRK lysis buffer. Centrifuge at 14,000 × g for 5 minutes at room temperature to remove any undissolved impurities. Carefully transfer the supernatant into a 1.5 mL centrifuge tube. 2) Add an equal volume of 70% ethanol to the lysate and mix thoroughly by vortexing or pipetting. 3) Place the column in a collection tube and transfer the mixture into the column. Centrifuge at 10,000 × g for 30–60 seconds, then discard the filtrate. 4) Transfer the column to a new collection tube, add 300 μL of RNA Wash Buffer I, centrifuge under the same conditions, and discard the filtrate. Return the column to the collection tube. 5) DNase Digestion: Prepare DNase Digestion Buffer (73.5 μL Digestion Buffer + 1.5 μL RNase-Free DNase I), mix well, and apply the mixture to the column membrane. Incubate at room temperature for 15 minutes. 6) Add 500 μL of RNA Wash Buffer I to the column, centrifuge, and discard the filtrate. 7) Add 500 μL of RNA Wash Buffer II to the column, centrifuge, and discard the filtrate. 8) Repeat step 7 with a new collection tube. 9) Dry the column by centrifuging it at 10,000 × g for 2 minutes. 10) Place the column on a 1.5 mL tube, add 30–100 μL of DEPC water, let stand for 2 minutes, and elute the RNA by centrifugation at 10,000 × g for 1 minute. 11) RNA Concentration Determination: Dilute the extracted RNA 10-fold with DEPC water. Measure the concentration using a Nanodrop spectrophotometer, with DEPC water as the blank. Record both the concentration and the OD260/OD280 ratio. **2. RNA Electrophoresis Detection** 1) Denatured Agarose Gel Preparation: Dissolve 1 g of agarose in 75 mL of deionized water and heat until fully dissolved. Cool to approximately 70°C, then add 10 mL of 10× MOPS and 15 mL of formaldehyde and EB. Pour the gel into a comb mold and allow it to solidify. 2) Electrophoresis Buffer Preparation: Dilute 50 mL of 10× MOPS to 500 mL with deionized water. Add EB to the running buffer. 3) RNA Sample Preparation: Take 3 μL of RNA, 2 μL of 10× MOPS, and adjust the volume to 20 μL with DEPC water. Incubate at 65°C for 10 minutes. Add 2 μL of 10× RNA loading buffer before electrophoresis. 4) Electrophoresis: Load the samples onto the gel and run at 100 V for about 5 minutes. Continue electrophoresis until the bromophenol blue reaches about two-thirds of the gel. **3. Reverse Transcription** 1) Prepare all reagents required for the reaction. Gently invert the mixture, briefly centrifuge, and keep on ice. 2) Prepare the RNA-Primer Mix (all on ice): - Total RNA: 1 μg - 60 μM Oligo(dT)18: 1 μL (final concentration: 2.4 μM) - DEPC water: Adjust to 13 μL total volume 3) Denature the RNA-Primer Mix by incubating at 65°C for 10 minutes, then quickly cool on ice. 4) Prepare the Reverse Transcription Reaction Mix: - RNA-Primer Mix: 13 μL - 5× RT Reaction Buffer: 5 μL (final concentration: 1×) - 25 mM dNTP: 1 μL (final concentration: 1 mM) - 25 U/μL RNase Inhibitor: 1 μL (final concentration: 1 U/μL) - 200 U/μL M-MLV RTase: 1 μL (final concentration: 8 U/μL) - DEPC water: 4 μL - Total volume: 25 μL 5) Incubate the reaction mix at 42°C for 1 hour after brief centrifugation. 6) Inactivate the enzyme by heating at 85°C for 5 minutes, then store the cDNA at -20°C. **4. Quantitative PCR (qPCR)** The relative quantification was performed using the SYBR Green I dye method based on the ΔΔCt analysis. The procedure is as follows: 1) Allow the 2X AllinOne™ Q-PCR Mix to reach room temperature. Gently invert the tube and briefly centrifuge. Keep away from light during use. 2) Prepare the qPCR reaction mix on ice: - 2X AllinOne™ Q-PCR Mix: 10 μL (final concentration: 1×) - ddH2O: 1 μL - Forward Primer (4 μM): 2 μL (final concentration: 0.4 μM) - Reverse Primer (4 μM): 2 μL (final concentration: 0.4 μM) - cDNA: 5 μL - Total volume: 20 μL *Note: A no-template control (NTC) was included as a negative control, where water replaced the cDNA.* 3) Briefly centrifuge the tubes to ensure the reaction mix is at the bottom of the wells. 4) Perform the qPCR using a standard three-step program: - Pre-denaturation: 95°C for 10 minutes - Denaturation: 95°C for 10 seconds - Annealing: 57°C for 20 seconds - Extension: 72°C for 15 seconds - Repeat for 40 cycles 5) After the PCR, perform a melting curve analysis: - Temperature range: 72°C to 95°C - Step size: 0.5°C per step - Time per step: 6 seconds - Final hold: 30°C for 30 seconds

Living Room Chairs

living room lounge chairs,Living Room Chairs,living room half recliner chairs

Guangzhou LoPhiDa Co.Ltd , https://www.gzwidinlsa.com