Recommended protocols for collecting and preparing biosamples for RNA extraction by Arraystar. If you use other protocols or methods, please provide us the details prior to your sample submission.
The following protocols are recommended for collecting and preparing biosamples for RNA extraction by us. If you use other protocols or methods, please provide us the details prior your sample submission. You may also use these protocols for your own RNA extraction and submit the RNA samples for the project to us.
RNA in cells, tissues and other sources of biosamples is very sensitive to RNase digestion and degradation. Collect and preserve samples as fast as possible for the best RNA quality. All RNA work must be carried out in a work area thoroughly decontaminated for RNases. Always use RNase-free labwares and reagents. Wear gloves and lab coat.
If your biosamples were potentially exposed to or contain any biohazardous agents, you must disclose before placing your order and shipping your samples. We will evaluate and determine the biosafety compliance and acceptance of the samples.
The recommended cell number per sample: 2 million cells
The cells should be immediately lysed in TRIzol. For cells, RNAlater is not recommended as it is less convenient. Storing and shipping unpreserved frozen cell pellet is not recommended. Cell culture contaminations, such as mycoplasma, chlamydia bacteria, and fungi, will impact your research results. Our sample QC doesn’t include any contamination testing. Please keep your valuable cells safe from any contamination during the culture and do the testing prior to the sample shipment.
1) Harvest cells by centrifugation. Do not wash the cells to minimize RNA degradation. 2) Immediately add to the cell pellet in 1 mL TRIzol Reagent per 5~10×106 cells. 3) Lyse the cells by repetitive pipetting up and down. 4) Store the lysate at -80°C. Ship on dry ice.
1) Aspirate away the culture medium. No need to rinse the cell monolayer to minimize RNA degradation. 2) Lyse cells directly in the dish by adding 1 mL TRIzol Reagent. The amount of TRIzol Reagent is based on the culture dish surface area (1 mL per 10 cm2) and not on the number of cells present. 3) Pass the cell lysate several times through a pipet tip. 4) Store the lysate at -80°C. Ship on dry ice.
To minimize the RNA degradation during sorting, the cells should be sorted directly into TRIzol-LS Reagent (for Liquid Sample). 1) Place 500 uL TRIzol-LS in the FACS tube. 2) Sort directly into the tube, mix as necessary. 3) After the sorting, measure the volume and calculate the sample volume from the sorting. 4) Add TRIzol-LS so that the (sample volume)/(total volume) ≤ 30%. 5) Proceed with the standard TRIzol-LS protocol for RNA extraction.
The recommended tissue amount (tissue mass by weight) per sample: 10~25 mg
RNAs preserved in TRIzol and RNAlater have similar RNA quality. For tissues, RNAlater is more convenient at the site of sample collection. TRIzol method is easier at homogenizing freshly collected tissues.
1) Excise the tissue sample within 10 minutes after death. Rinse in PBS and blot dry briefly. Cut it into slices less than 5 mm thick. Perform this step as quickly as possible and proceed immediately to Step 2. 2) Completely submerge the tissue piece(s) in the collection vessel containing RNAlater RNA Stabilization Reagent. At least 10 volumes of the reagent (or approximately 10 uL reagent per 1 mg of tissue) is required. 3) Incubate the tissue in the reagent at 2~8°C overnight. 4) Store at -20°C. 5) Ship on ice.
1) Excise the tissue sample within 10 minutes after death. Rinse in PBS and blot dry briefly. Cut the tissues into small pieces to ensure that the tissue is completely immersed in TRIzol reagent. Transfer tissue pieces in 1 mL TRIzol Reagent per 10~25 mg tissue. The tissue volume should not exceed 10% of the TRIzol Reagent volume. 2) Homogenize the tissue in TRIzol on ice using a tissue homogenizer. 3) The lysate can be stored at -4°C for days, -20°C for a year or ~ -80°C indefinitely. 4) Ship on dry ice.
1) Wash the fresh tissue with 1× PBS and blot dry briefly. If possible, cut the tissue into small pieces. Perform this step as quickly as possible and proceed immediately to step 2. 2) Freeze the tissue in liquid nitrogen for 2~3 hours. 3) Store at -80°C or in liquid nitrogen. 4) Ship on dry ice. Alternatively, grind or pulverize the tissue samples to powder in liquid nitrogen, followed by homogenization in TRIzol, and shipped at 4°C or on dry ice.
1) The quality of RNA isolated from FFPE samples will depend on the technique used to preserve the sample and on other factors such as the age of the block. 2) The standard fixation protocol below is best suited for gene expression analysis: fixate tissue sample in 4-10% formalin immediately after surgical removal; use a fixation time of 14-24 hours; completely dehydrate the samples prior to embedding in paraffin. 3) Ship at room temperature.
| Biofluid | Starting Vol for Services Without IP or Pretreatment | Starting Vol for Services With IP or Pretreatment | Starting Vol for Exosome Isolation |
|---|---|---|---|
| Whole blood | 1~2 mL (0.3 mL/aliquot) | 2~3 mL (0.3 mL/aliquot) | – |
| Plasma | 1~2 mL (0.3 mL/aliquot) | 2~5 mL (0.3 mL/aliquot) | 2 mL |
| Serum | 1~2 mL (0.3 mL/aliquot) | 2~5 mL (0.3 mL/aliquot) | 2 mL |
[1] Biofluid RNA yields typically vary greatly among individuals. The actual yields can differ very largely from these numbers. All the volumes here are just for reference only. [2] IP or Pretreatment: Pretreatment for tRNA, tRF&tiRNA Seq/PCR Array Services. [3] We do not provide exosome isolation service. We accept isolated exosomes for RNA purification. Isolated exosome samples shall be lysed in TRIzol and shipped on dry ice.
1) Use serum tubes to collect whole blood. Separate serum from the clot within 2 hours. 2) Gently invert the tube 4~5 times. Stand vertically at room temperature until fully clotted (~ one hour). 3) Separate the serum by centrifugation at 1000~1300g for 10 minutes. 4) Transfer the serum to ~300 uL aliquots. 5) For short time storage, -20~-80°C is OK. For long term storage, use liquid nitrogen. 6) Ship on dry ice.
1) Use EDTA or citrate based anticoagulant tubes to collect whole blood. DO NOT use heparin as the anticoagulant, as RNA bound heparin is difficult to remove and interferes with enzymatic reactions. 2) Immediately separate cells and plasma by centrifugation at 1000~1300g for 10 minutes. 3) Transfer the plasma to ~300 uL aliquots. 4) For short time storage, -20~-80°C is OK. For long term storage, use liquid nitrogen. 5) Ship on dry ice.
1) Use anticoagulant tubes to collect whole blood. 2) Transfer the blood into ~300 uL aliquots. 3) For short time storage, -20~-80°C is OK. For long term storage, use liquid nitrogen. 4) Ship on dry ice.
1) Use anticoagulant tubes to collect 5 mL whole blood. 2) Add 9 mL red blood cell lysis solution (e.g. Red Blood Cell Lysis Buffer, Sigma-Aldrich) to 3 mL blood. Gently invert the tube several times. 3) Let the tube stand for 5~10 minutes at room temperature. 4) Centrifuge at 2500g for 5 minutes and remove the supernatant. 5) Resuspend the cells in 1 mL PBS. 6) Repeat red blood cells lysis. 7) Let the tube stand for 5~10 minutes at room temperature. 8) Centrifuge at 2500g for 5 minutes and remove the supernatant. The white cell purity is > 80%. 9) Lyse the cells in TRIzol (5 million cells per 1 mL TRIzol). Pipet up and down until complete lysis. 10) Aliquot ~500 uL per tube for shipment on dry ice.
1) Use sufficient input volume and exosome isolation kit capacity to isolate the exosomes by the method of your choice. 2) Resuspend the exosomes in 200 uL PBS and mix with 800 uL TRIzol Reagent (v:v = 1:4). 3) Ship on dry ice.
Please complete, sign, and email us a Project Form prior to sending your samples. The sample package shall be shipped to the address:
ATTN: Samples Receiving (Project#______ )
Arraystar Inc.
9430 Key West Avenue #128
Rockville, MD 20850 USA
Tel: 888-416-6343
All shipments and inquiries should reference the Project Number assigned to your project by Arraystar. You will receive notification from us upon receipt of your samples. We’ll notify you if there are any discrepancies between your Sample Submission Form and the contents we receive, or if there are any issues with the physical condition of the samples.
For the best results, please provide sufficient-amount and high-quality biosamples to us. If the RNA extracted from your biosamples couldn’t meet the amount and quality requirements, we will contact you for you to decide whether to replace, proceed, or drop the sample from the study. With your consent and approval, it is possible for us to proceed with the experiment. In such a case, you understand the data quality and the success rate may decrease progressively with the lowered sample amount or quality. Although usable data may still be obtainable, we cannot predict for certainty nor do we guarantee the quality and success rate of the data outcomes.
Common questions about collecting and shipping biosamples
Contact our technical support team for sample amount and quality questions, or request a quote for your tRNA and tRF&tiRNA project.