Kit for in-vitro assisted diagnosing pancreatic cancer
A diagnostic reagent and kit technology, applied in the field of kits for the in vitro auxiliary diagnosis of pancreatic cancer, can solve the problems of inability to be used as an indicator for the diagnosis of pancreatic cancer, misdiagnosis, lack of human experience, etc.
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Embodiment 1
[0044] Example 1 Sample requirements and sample processing methods
[0045] 1. Sample requirements
[0046] 1) Sample type: serum.
[0047] Serum should be freshly collected and separated. Within 6 hours of blood collection, the serum must be separated, collected, and transferred to a single-use RNase-free sterile microcentrifuge tube.
[0048] 2) Serum samples can be stored stably at room temperature for 24 hours, at 2°C-8°C for 7 days, and below -18°C for 6 months.
[0049] 3) The RNA solution extracted from serum samples can be stored stably for 12 hours at room temperature, 3 days at 2°C-8°C, and 3 months below -18°C.
[0050] 2. Processing method:
[0051] 1) Process the whole blood sample into serum: collect 2ml of venous blood and put it in a 5ml clean centrifuge tube without anticoagulation. After standing still for 30 minutes, centrifuge at 5000 rpm for 5 minutes, and take the supernatant. If the sample is not used immediately, store it below -18°C. The serum s...
Embodiment 2
[0053] Embodiment 2 reverse transcription operation process
[0054] 1. The reverse transcription system is shown in Table 4:
[0055] Table 4
[0056] name
volume
RT buffer
2μl
0.5μl
dNTPs
1μl
Reverse transcription primers (RP-miR-25, RP-internal control)
0.5μl
2μl
DEPC water
4μl
total capacity
10μl
[0057] 2. Experimental operation:
[0058] 1) Calculation of the reaction system: Calculate the amount of the system required for the reverse transcription reaction of miR-25 and the internal control according to the test sample size.
[0059] 2) Prepare the reaction mixture: add DEPC water, RT buffer, dNTPs, reverse transcriptase, reverse transcription primers (RP-miR-25, RP-internal control) in sequence, and mix well.
[0060] 3) Distributing the reaction mixture: distribute 8 μl / well of the mixture obtained in step 2) in corresponding 96-well PCR plat...
Embodiment 3
[0066] Embodiment 3PCR operation process
[0067] 1. The experimental system is shown in Table 5:
[0068] table 5
[0069] name
volume
qPCR buffer
2μl
MgCl 2
1.2μl
dNTPs
0.4μl
0.3μl
Primer probe (PP-miR-25, pp-internal control)
0.5μl
ROX
0.2μl
cDNA
5μl
DEPC water
10.4μl
total capacity
20μl
[0070] 2. Experimental operation:
[0071] 1) Calculate the reaction system: Calculate the amount of system required for each qRCR primer according to the experimental requirements.
[0072] 2) Prepare the reaction mixture: add DEPC water, qRCR buffer, MgCl2, dNTPs, DNA polymerase, ROX, primer probe (PP-miR-25, PP-internal control) in order, and mix well.
[0073] 3) Distributing the reaction mixture: distribute 15 μl / well of the mixture obtained in step 2) in corresponding 96-well PCR plates.
[0074] 4) Add cDNA: Add 5 μl / well of the sample cDNA ob...
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