Please use a delivery service such as FedEx, DHL or UPS for all samples. A timely notice must be sent by mail before sending your samples, which includes the Sample Submission Form and the tracking number (for timely registration and processing after the arrival of the samples). Make sure your sample arrives on weekdays.
RNA sample should be submitted in RNase-free water, RNA Stabilization Reagent, or 10mM Tris pH 8.0. All total RNA samples should be DNA-free. RNA samples require an OD A260/A280 ratio ≥ 1.8, A260/230 ratio≥ 1.8 and a RIN ≥ 6. Ship with dry ice.
Take fresh tissues, the tissue volume should be small, try to length, width and height ≤ 0.5cm, considering the operability, the size of the cut tissue block can refer to the size of green bean particles.
Remove non-studied tissue types (e.g. connective tissue, adipose tissue, etc.), if it is a clinical lesion tissue collection to correctly determine the lesion as well as normal tissue, the normal tissue around the lesion tissue should be removed, and vice versa.
Quickly wash the blood and stains on the surface of the tissue with 1×PBS or saline prepared with pre-cooled RNase-Free water at 2-6°C, and use dust-free paper towels to absorb the liquid on the surface.
The treated tissues were quickly put into pre-cooled RNase-Free numbered ultra-low temperature lyophilization tubes with threaded mouths that were resistant to -192°C, quickly placed in liquid nitrogen snap freeze for 1 hour and then transferred to -80°C for long-term storage, avoiding repeated freeze-thawing before RNA extraction.
Cell Sample
Adherent Cells
Remove the wall-grown cells from the incubator, observe the cells under the microscope to determine the good growth state and discard the medium.
Carefully add 1×PBS prepared with enzyme-free water in the same volume as the culture medium, wash the cells for 1 min, then discard the PBS and repeat once.
Add the appropriate amount of lysis solution and repeatedly blow until full lysis (TriZol for example, a large dish of 10-15cm, add 1-2 mL each time, blow and mix well, the criteria for full lysis are that the liquid is not viscous when blowing, good fluidity. If the liquid is too viscous, it means that the lysis solution is not sufficient, and it is necessary to add the lysis solution.
Transfer to a threaded mouth lyophilization tube that is resistant to -192°C and then store in -80°C refrigerator for long term storage and transport on dry ice.
Suspension Cells
Select the suspension cell in good growth state.
Centrifuge at 200-1000 g for 5-10min to obtain cell precipitate and discard the medium.
After adding the appropriate amount of RNase-Free water prepared with 1×PBS, resuspend the cell precipitate by gently blowing with a wide muzzle gun, then centrifuge at 200 g for 5min, discard the PBS, and repeat once.
Add appropriate amount of TriZol lysis solution and blow repeatedly until fully lysed (lysis criteria are similar to the above treatment of walled cells).
Transfer to a -192°C threaded mouth lyophilization tube and store at -80°C for long term storage and transport on dry ice.
Plant Tissue Sample
The younger and fresher the plant tissue, the less secondary metabolites it contains. As the plant matures, it will contain more and more secondary metabolites, which will affect RNA extraction.
(Optional) Quickly wash the tissue surface with pre-chilled RNase-Free water prepared with 1×PBS or saline to stain the surface and blot up the liquid. Cut into appropriate size with scissors, preferably no longer than 2 cm if not exceptional.
The treated tissues are quickly put into pre-cooled RNase-Free threaded lyophilization tubes that have been written with a number to resist -192°C, or wrapped in tin foil marked with a name and snap frozen in liquid nitrogen for >1 h. Transfer to -80°C for long-term storage, avoid repeated freeze-thawing before RNA extraction, and send samples on dry ice. If the sample is not cooled down by liquid nitrogen flash freezing 10 min after isolation, it is very likely to cause sample degradation. Do not store the sample directly at -80°C, as the low activity of RNase can still cause degradation of the sample.
Body Fluid Sample
Use vacuum blood collection tubes to collect whole blood and separate the serum, or use EDTA purple anticoagulation tubes to collect whole blood samples and separate subsequent plasma samples. The use of heparin tubes for whole blood collection is not recommended because heparin can affect the efficiency of reverse transcription of PCR enzymes.
Whole Blood Sample Collection
Before blood collection, the blood collection tube should be brought back to room temperature, the recommended temperature is 18-25°C.
When collecting blood, first collect 1-2mL of blood sample with another blood collection tube, discard that blood collection tube and then use blood collection tube. This effect is because the blood collector volume can be pre-filled, another to reduce the impact caused by blood pressure, a certain degree of cleaning the blood collection tube.
After blood collection, immediately mix up and down upside down about ten times, 18-25°C ortho-statically placed for 2h.
If not extracted immediately, place the blood collection tube upright on a plastic test tube rack, first at -20°C for 24 h, and then transfer to -80°C for long-term storage or transfer to dry ice for transportation. Transfer the whole blood sample from the blood collection tube to a threaded-mouth lyophilization tube and place it at -80°C without flash-freezing the tube in liquid nitrogen for 1 h.
Please use thick-walled foam boxes with sufficient dry ice for transport and avoid repeated freezing and thawing during sample storage and transport.
Peripheral Blood Mononuclear Cells (PBMC) Isolation From Plasma
Follow up the step of centrifugation of EDTA anticoagulation tubes in the previous step, and then restore Ficoll and PBS from low temperature to room temperature.
Add 10mL of Ficoll to a 50mL centrifuge tube (labeled A), and then preferably leave it for a period of time; add 10mL of PBS to another 50mL centrifuge tube (labeled B).
Dilution: Gently shake the anticoagulated blood collection tube, remove the cap with kimwipes or sterile gauze and set aside, transfer 10mL of blood to centrifuge tube B, and mix gently to obtain PBS mixture (20 mL).
Moderately tilt centrifuge tube A and add 20mL of PBS mixture slowly along the wall (be careful to go slow, too fast will penetrate the Ficoll layer and greatly affect the extraction effect).
Gradient centrifugation: carefully place the tube into the centrifuge (do not disturb the liquid layer), centrifuge at room temperature 400 g for 20min.
After centrifugation, carefully remove it and place it on the operating table, visible in four layers.
Optional: remove 2-3 mm of plasma from the lymphocyte layer first to facilitate aspiration later.
Pipette as much PBMC as possible with the P1000 and transfer to a 15mL centrifuge tube, avoiding plasma and Ficoll as much as possible.
Add PBS to make the volume to 10 mL, cover and gently turn over and shake well 5 times. Remove supernatant and flick the end of the tube until the cell mass is completely resuspended in the remaining PBS.
Collect whole blood using a vacuum blood collection tube, etc.
After mixing ten times by gently inverting the top and bottom, immediately place the whole blood in an upright position at 4°C or in an ice box.
Centrifugation at 1800 g for 10min to obtain the upper serum sample (whole blood needs to be separated within 1 h, according to the serum tube operation instructions or the customer's laboratory serum separation procedure).
Transfer the serum to a 1.5 mL centrifuge tube and centrifuge at 13,000g for 2min.
Transfer the supernatant to the specification in 200 μL-1.5 mL -192°C ultra-low temperature resistant threaded mouth lyophilization tube, at least ≥100μL (0.1mL) per sample. Store in liquid nitrogen for 1 h at -80°C and transport on dry ice.
It is recommended to use purple EDTA anticoagulation tubes to collect whole blood samples.
After mixing ten times by gently inverting the top and bottom, immediately place the whole blood in an upright position at 4°C or in an ice box.
Centrifuge at 1200 g for 10min to obtain the upper plasma sample (whole blood needs to be separated within 1h, according to the anticoagulation tube operation instructions or the customer's laboratory plasma separation procedure).
Transfer the plasma to a 1.5mL centrifuge tube and centrifuge at 13000 g for 2min.
Transfer the supernatant to a 200 μL-1.5 mL -192°C ultra-low temperature threaded mouth lyophilization tube with at least ≥100 μL (0.1 mL) per sample. Store in liquid nitrogen for 1h at -80°C and transport on dry ice.
Collect cell supernatant, fresh urine, saliva, amniotic fluid, cerebrospinal fluid, making sure not to hemolymph (blood proteins can contaminate sample proteins). Place at 4°C or in an ice box in an upright position and transfer to RNase-free centrifuge tubes. The following operations should be performed within 1 h after sample collection to prevent RNA degradation.
Centrifuge at 3000g for about 10 min at 4°C to remove cells and debris.
Pour the supernatant carefully into a new RNase-free tube (be careful not to transfer the precipitate) and centrifuge at 13,000g for 2 min at 4°C to remove residual debris and cells.
Transfer the supernatant to a new -192°C ultra-low temperature threaded mouth lyophilization tube, snap freeze in liquid nitrogen for 1 h (optional) and seal tightly. Store at -80°C and transport on dry ice.
Paraffin Tissue Samples Collection
A single extraction of RNA requires an amount of 35 mg or more. The total amount of surgical tissue cut from the pathology department ≥ 35mg (green bean size, not to be taken from the section part of the tissue exposed to air), biopsy tissue length > 1 cm, 2-3 strips required, wax block intact, no bump damage to the paraffin-embedded tissue. Unsliced paraffin blocks can be transported at 4°C or room temperature.
Microbial Sample
Non-Pathogenic Bacterial Collection
The number of bacteria required for one reaction ≤ 1×107
Observe the growth status under microscope and collect the bacteria in logarithmic growth phase.
Transfer the appropriate volume of bacterial solution to a 2mL screw cap sharp bottom RNase-Free centrifuge tube and centrifuge at 14000 g for 4-10 min at room temperature or 4°C.
Discard the medium, put the bacterial precipitate quickly in liquid nitrogen flash freezing >1h, then transfer to -80°C or liquid nitrogen for long-term storage, dry ice transport.
Non-Pathogenic Fungi Collection
The amount of fungi required for one reaction is ≤50mg, weigh the fungi and divide them into multiple tubes; place the weighed fungi in pre-cooled ultra-low temperature resistant -192°C threaded lyophilization tubes, quickly put into liquid nitrogen, quick-freeze for >1h, transfer to -80°C for long-term storage or send directly with dry ice.
Pathogenic Sample
All samples of potentially pathogenic fungi and bacteria need to be pretreated. Please contact us for specific operations.
* For Research Use Only. Not for use in diagnostic procedures.
