|
一、RNA Extraction
1、Sample Lysis:
Tissue Samples:
Trizol Volume: Add 1 mL Trizol per 50-100 mg tissue. Sample volume should not exceed 10% of Trizol volume.
(1) Mince animal or plant tissues into small pieces, grind in liquid nitrogen, or homogenize using a homogenizer.
(2) Transfer the ground tissue powder into a 2 mL centrifuge tube containing 1 mL Trizol. Vortex vigorously to mix, then place on ice until all samples are processed.
(3) The lysate should appear as a clear, viscous liquid. For protein-, fat-, or polysaccharide-rich tissues (e.g., muscle, adipose tissue, plant nodules), insoluble material may remain. Centrifuge at 4°C, 12,000 × g for 10 min, then transfer the supernatant to a new tube.
Cell Samples:
(1) Adherent cells: Remove the culture medium. For every 10 cm² culture area (e.g., a well in a 6-well plate or a 35 mm dish), add 1 mL of Trizol. Use a pipette to repeatedly pipette up and down to ensure complete cell lysis, then transfer to a centrifuge tube. Note: The volume of Trizol should be determined by the culture area, not the cell number. Insufficient Trizol may lead to DNA contamination in the extracted RNA.
(2) Suspension cells: Centrifuge to collect cells and remove the liquid. For every 5-10×10⁶ animal, plant, or yeast cells, or every 1×10⁷ bacterial cells, add 1 mL of Trizol. Use a pipette to lyse the cells completely, then transfer to a centrifuge tube. Note: Do not wash the cells before adding Trizol to prevent RNA degradation. Use a homogenizer if necessary to lyse certain bacterial or yeast cells.
2、Phase Separation:
(1) Incubate the lysate at room temperature for 5 min to allow complete separation of nucleoprotein complexes. (Note: The samples can be stored at -80℃ for long-term preservation at this point.)
(2) For every 1 mL of Trizol used, add 0.2 mL of chloroform. Close the tube tightly, shake vigorously for 15 s, and incubate at room temperature for 2-3 min.
(3) Centrifuge at 4℃ and 12,000 x g for 15 min. The sample will separate into three layers: a yellow lower organic phase, an intermediate phase, and a colorless upper aqueous phase.
(4) Transfer the upper aqueous phase containing total RNA to a new centrifuge tube. The volume of the aqueous phase should be approximately 60% of the initial Trizol volume.
3、RNA Recovery:
(1) For every 1 mL of Trizol used initially, add 0.5 mL of isopropanol. Invert several times to mix well and incubate at room temperature for 10 min.
(2) Centrifuge at 4℃ and 12,000 x g for 10 min. Discard the supernatant, and a gel-like RNA precipitate will be visible.
(3) For every 1 mL of Trizol used initially, add 1 mL of 75% ethanol. Invert several times to wash the precipitate.
(4) Centrifuge at 4℃ and 12,000 x g for 5 min. Discard the supernatant.
(5) Air-dry the pellet by inverting the tube at room temperature for 5-10 min or use a vacuum (but avoid a vacuum centrifuge to prevent over-drying of RNA, which may make it difficult to resuspend).
(6) Dissolve the RNA in an appropriate volume (e.g., 25 μL) of DEPC-treated water or TE buffer by pipetting up and down.
(7) Determine the concentration, purity, and integrity of RNA using agarose gel electrophoresis and a UV spectrophotometer.
(8) Use the RNA immediately or aliquot and store at -80℃ to avoid repeated freeze-thaw cycles.
Product Recommendations:
| Catalog No. |
Product Name |
Specification |
|
abs60154
|
Trizol |
100mL/500mL
|
|
abs9331
|
Trizol (Total RNA Extraction Kit) |
1kit
|
|
abs9259
|
DNase/RNase-Free Water |
500mL
|
二、Reverse Transcription
1、Removing Residual Genomic DNA from RNA:
| Component |
Volume |
| RNA Template |
≤ 1 µg total RNA or ≤ 0.1 µg poly(A) mRNA |
| 5×gDNA Remover Buffer |
2 µL |
| gDNA Remover |
1 µL |
| Nuclease-Free Water (DEPC-treated) |
To a final volume of 10 µL |
Mix and briefly centrifuge. Reaction conditions: 37℃, 5 min.
When using your own sequence-specific primers, the amount of RNA template can be adjusted to “≤ 5 µg total RNA or ≤ 0.5 µg poly(A) mRNA”.
2、Directly add the following components for reverse transcription reaction to the above tube to perform the first-strand cDNA synthesis:
| Component |
Volume |
|
gDNA remover-treated RNA sample
|
10 uL
|
|
5 x Buffer (with primer)
|
4 uL
|
|
Enzyme mix
|
1 uL
|
|
Nuclease-free Water (DEPC-treated)
|
To a final volume of 20 µL
|
3、Gently mix and briefly centrifuge; incubate at 42℃ for 15-50 min. Note: For complex templates, the reverse transcription temperature can be increased to 50℃ to improve the efficiency of reverse transcription. The reaction time can be adjusted appropriately according to the experimental application scenario. If the synthesized cDNA is used as a template for qPCR, the reaction conditions are 15 min incubation at 42℃.
4、Heat at 85℃ for 5 min to inactivate the Enzyme mix.
5、After the reaction, the obtained cDNA should be placed on ice for subsequent experiments or stored at -20℃.
Product Recommendations:
| Catalog No. |
Product Name |
Specification |
|
abs601510
|
First-strand cDNA Synthesis Mix With gDNA Remover
|
50T/100T/200T
|
三、qPCR Experiment
Dye-based qPCR (using product abs601511 as an example)
Reagents to be provided by the user: cDNA or DNA template, primers, ROX Reference Dye/ROX Reference Dye II.
Please follow the instructions for use according to the specific brand of the real-time quantitative PCR instrument.
Operation example: Taking 20 µL and 50 µL PCR reaction systems as examples:
1、Establishment of PCR Reaction System:
| Component |
20µL System |
50µL System |
|
DNA Template
|
1uL
|
1uL
|
|
Forward Primer (10µM)
|
0.5uL
|
1uL
|
|
Reverse Primer (10µM)
|
0.5uL
|
1uL
|
|
2×RealStar Green Fast Mixture
|
10uL
|
25uL
|
|
ROX Reference Dye/ROX Reference Dye II
|
0.4uL
|
1uL
|
|
RNase-free H2O
|
To a final volume of 20µL
|
To a final volume of 50µL
|
Note:
-
Template amount: Use 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference. Due to the varying copy numbers of target genes in templates from different species, gradient dilution of the template may be performed to determine the optimal template usage. Additionally, when using cDNA (RT reaction mixture) from a Two Step RT-PCR reaction as a template, the volume added should not exceed 10% of the total PCR reaction volume.
-
Primers: Typically, a primer concentration of 0.2 μM yields good results, with a final concentration range of 0.1-1.0 μM as a reference for setting. If amplification efficiency is not high, increase the primer concentration; if nonspecific reactions occur, decrease the primer concentration to optimize the reaction system. To achieve ideal qPCR results, it is recommended that the amplification fragment length be 80-200 bp.
-
Different instruments require different ROX Reference Dye, or it may be necessary to add or not add it. Please follow the instrument instructions for operation.
2. Setting of PCR reaction conditions: The HotStart Taq DNA Polymerase used in this product is a HotStart DNA polymerase blocked by anti-Taq antibodies. If pre-denaturation of the template is performed before the PCR reaction, it is usually set to 95℃ for 2 min, with a proper extension of time up to 5 min for complex or high GC templates. This DNA polymerase can complete amplification of at least 300 bp within 15 s, which can meet the needs of most qPCR experiments; for amplicons exceeding 350 bp or with high GC content, it is recommended to increase the extension time to 60 s or use a three-step method to improve amplification efficiency.
|
Two-step PCR Amplification Standard Program:
|
|
Predenaturation
|
95℃
|
2min
|
40 Cycles
|
|
Denaturation
|
95℃
|
15s
|
|
Annealing/Extension
|
60℃
|
15-30s
|
|
Melting Curve (automatically set by the instrument)
|
|
Theer-step PCR Amplification Standard Program:
|
|
Predenaturation
|
95℃
|
2min
|
40 Cycles
|
|
Denaturation
|
95℃
|
15s
|
|
Annealing
|
60℃
|
15-30s
|
|
Extension
|
72℃
|
30s
|
|
Melting Curve (automatically set by the instrument)
|
Note: The example provided is a standard qPCR reaction system and is for reference only. Actual reaction conditions may vary depending on the structure of the template, primers, etc. Optimal reaction conditions should be set based on the characteristics of the template, primers, and target fragment, and the reaction system should be scaled up or down accordingly.
3. Complete the experiment on the corresponding Real Time PCR instrument and analyze the results.
Product Recommendations:
| Catalog No. |
Product Name |
Specification |
Application |
|
abs601511
|
2× Premixed Real-Time Quantitative PCR Fast Reaction System
|
1.7mL×3
|
Dye-based qPCR
|
|
abs601512
|
2× Premixed Real-Time Quantitative PCR Fast Reaction System (High Concentration ROX)
|
1.7mL×3
|
Dye-based qPCR
|
|
abs601513
|
2× Premixed Real-Time Quantitative PCR Fast Reaction System (Low Concentration ROX)
|
1.7mL×3
|
Dye-based qPCR
|
|
abs60305
|
TaqMan Real-Time Quantitative PCR Kit
|
100T/200T/500T
|
TaqMan qPCR
|
|
abs60307
|
TaqMan Super Multiplex Real-Time Quantitative PCR Kit
|
100T/200T/500T
|
TaqMan qPCR
|
耗材产品推荐：
| Catalog No. |
Product Name |
Specification |
|
abs7089
|
0.1mL PCR Reaction Plate (Transparent, Half-Skirted)
|
1 box
|
|
abs7090
|
0.1mL PCR Reaction Plate (Transparent, Non-skirted)
|
1 box
|
|
abs7091
|
0.2mL PCR Reaction Plate (Transparent, Half-Skirted)
|
1 box
|
|
abs7092
|
0.2mL PCR Reaction Plate (Transparent, Non-skirted)
|
1 box
|
|
abs7246
|
Real-Time PCR Sealing Film (Pressure-sensitive, Strong Adhesion)
|
1 box
|
|
abs7247
|
Real-Time PCR Sealing Film (24/48 Wells, Pressure-sensitive)
|
1 box
|
|
abs7248
|
PCR Sealing Film (Strong Adhesion, High Transparency)
|
1 box
|
|
abs7093
|
100µL PCR 8-tube Strip (With Cap)
|
1 box
|
|
abs7094
|
200µL PCR 8-tube Strip (With Cap)
|
1 box
|
|
abs7119
|
1.5mL Centrifuge Tube (Sterile, RNase-free)
|
1 box
|
|
abs7118
|
0.5mL Centrifuge Tube (Sterile, RNase-free)
|
1 box
|
|
abs7071
|
10µL Filtered pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7074
|
200µL Filtered pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7077
|
1000µL Filtered pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7176
|
20µL Filtered pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7072
|
10µL pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7073
|
10µL pipette tip (Bagged, Sterile, RNase-free)
|
1 box
|
|
abs7075
|
200µL pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7076
|
200µL pipette tip (Bagged, Sterile, RNase-free)
|
1 box
|
|
abs7078
|
1000µL Long pipette tip (Boxed, Sterile, RNase-free) |
1 box
|
|
abs7079
|
1000µL pipette tip (Boxed, Sterile, RNase-free)
|
1 box
|
|
abs7080
|
1000µL pipette tip (Bagged, Sterile, RNase-free)
|
1 box
|
|
abs7081
|
1000µL Long pipette tip (Bagged, Sterile, RNase-free)
|
1 box
|
|
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