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Biosamples for RNA Extraction

Cells Tissues Biofluids Exosomes


Table of Contents

Biosafety. 2

RNA Extraction from Cells. 2

Cell Amount Requirement. 2

A. Suspension cells. 3

B. Adherent cells. 3

C. FACS sorted cells. 3

RNA Extraction from Tissues. 3

Tissue Amount Requirement. 3

A. RNAlater Method. 4

B. TRIzol Method. 4

C. Fresh frozen in liquid nitrogen. 4

D. FFPE samples. 4

RNA Extraction from Biofluids or Exosomes. 5

Biofluid Amount Requirement. 5

A. Serum collection. 5

B. Plasma collection. 6

C. Whole blood collection. 6

D. White blood cells from whole blood. 6

E. Exosome samples. 6

Sample Shipment. 6

Shipping on dry ice. 7

Sample Receipt Confirmation


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.

 

 

Our labs handle biosamples at Biosafety Level 2 (BSL    2) or below. Currently we do not accept samples potentially having biohazardous agents above BSL2 [Biosafety, 5th Edition].

 

 

Cell Amount Requirement

The recommended cell number per sample is 2×106 cells.
For MeRIP-seq requiring more RNA, the cell number is 1~2×107 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 macoplasma, chilamydia 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.

 

A. Suspension cells
    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.
B. Adherent cells
    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.
C. FACS sorted cells
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.

 

 

Tissue Amount Requirement

The Recommended tissue amount per sample is 10~25 mg tissue mass.
For MeRIP-sequencing requiring more RNA, the recommended tissue amount per sample is > 50 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.

 

A. RNAlater Method
    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.
B. TRIzol Method
    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.
C. Fresh frozen in liquid nitrogen
    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.
D. FFPE samples
    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:
        1))    Fixate tissue sample in 4-10% formalin immediately after surgical removal.
        2))    Use a fixation time of 14-24 hours.
        3))    Completely dehydrate the samples prior to embedding in paraffin.
    3)  Ship at room temperature.

 

Biofluid Amount Requirement[1]

Biofluid

Starting Vol for
RNA Extractio

Starting Vol for
Exosome Isolation[3]

Whole blood

1~2 mL
(0.3 mL/aliquot)

-

Plasma

1~2 mL
(0.3 mL/aliquot)

2mL

Serum

1~2 mL
(0.3 mL/aliquot)

2mL

Saliva

1~2 mL
(0.3 mL/aliquot)

-

Cerebrospinal fluid (CSF)

1~2 mL
(0.3 mL/aliquot)

-

Amniotic fluid

2~4 mL
(0.3 mL/aliquot)

-

Urine[2]

≥50mL

-

Cell culture media supernatant

-

50mL

NOTE:

[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] We do not provide urine RNA extraction service.
[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.

 

A. Serum collection
    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 temperate 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.
B. Plasma collection
    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.
C. Whole blood collection
    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.
D. White blood cells from whole blood
    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 by adding 9 mL red blood cell lysis solution. Gently invert the tube several times.
    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.
E. Exosome samples
    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

 

Shipping on dry ice

     Use screw-cap tubes as the containers; Snap-cap tubes may pop open during shipment. Seal the cap with Parafilm strip.

     Place the sample tubes in a plastic bag;

     Use 10 kg dry ice as refrigerant (sufficient for both domestic and international);

     Include a copy of the signed Project Form in a waterproof bag, separate from your samples;

     Check the carrier for dry ice/international shipping requirements;

     If possible, avoid a drop-off date with coming weekends, holidays, such as Thursday or Friday;

     Obtain the tracking number and notify by email the Arraystar representative who is working with you for the project.

 

Sample Receipt Confirmation

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.