RNA Preparation, QC and Shipping for RNA-Seq Applications

Transcription

1 Large-scale targeted NGS sequencing made easy & affordable. RNA Preparation, QC and Shipping for RNA-Seq Applications IMPORTANT NOTES!!! Wear gloves and maintain an RNase-free bench area for RNA isolation. STRONGLY ENCOURAGED on-column DNase treatment (e.g. RNase-Free DNase Set: Qiagen 79254) DO NOT use DEPC-treated H2O to dissolve RNA. Use RNase-free H2O supplied by Ambion, Qiagen, or other equivalent providers. Perform QC to assess the integrity and purity of your total RNA samples. Recommendations are provided below. See Table 1 below for submission mass guidelines. Follow the instructions for sample preparation, storage, and shipping Contact [email protected] when unsure. Sample submission requirements I. Recommended amount and quality of RNA samples for RNA-Seq Total Mass Conc. RIN Sample Submission Recommendations rrna ratio small/micro RNA-seq 2-5μg total RNA 50ng/μl OD260/OD ng small RNA 20ng/μl n/a n/a polya RNA-seq 20ng-1ug total RNA* 100ng/μl ultralow polya RNA-seq random-primed RNA-seq or stranded RNA-seq IP'ed, viral, pre-enriched RNA-seq 100pg-20ng total RNA* 2ng/μl ng total RNA 50ng/μl ng RNA 20ng/μl n/a n/a cdna 0.2-5μg dscdna 20ng/μl n/a n/a *Samples submitted at less than 2ng total mass or < 25,000 cells for extraction will not be QC tested upon receipt/extraction and data quality cannot be guaranteed. II. DNA Compatibility with RNA-Seq cdna constructed from the following kits is compatible with subsequent library preparation and NGS sequencing. SMARTer PCR cdna Synthesis Kit (Clontech Cat# & ) SMART-Seq v4 Ultra low Input RNA kit for Sequencing (Clontech Cat#634888, , , , , , ) TruSeq small RNA preparation kit (Illumina Cat# RS ) TruSeq stranded RNA preparation Kit (Illumina Cat# RS ) Kits that allow generation of ds cdna without Illumina sequencing adaptors Winters Chapel Rd., #100, Atlanta, GA USA [email protected]

2 III. Recommended RNA Preparation Methods for RNA-Seq Application A. Starting Materials Cultured Cells and Fine Needle Aspiration Biopsies (FNAB) Tissues Whole Blood Formalin-Fixed Paraffin-Embedded (FFPE) Laser Capture Microdissection (LCM) Flow Sorted Cells (FACS) contact Otogenetics for special requirements Other Biological Samples B. Total RNA Isolation Recommendations: High quality total RNA must be prepared using one of the following methods, each specific for your biological material. RNA material should be stored and shipped in dry ice to Otogenetics or lyophilized in RNAstable. Cultured Cells: Qiagen RNeasy Mini Kit or equivalent total RNA isolation kit with on-column DNase (RNase-free) treatment. Tissue Samples: Extract RNA from the tissue using a Trizol-based method with at least 10x volume of the Trizol reagent to that of the tissue volume. After the ethanol precipitation step, clean up further using Qiagen RNeasy Mini Kit or equivalent with oncolumn DNase treatment. FFPE Samples: Qiagen RNeasy FFPE Kit or equivalent. Samples Yielding Less than 100 ng Total RNA: Life Technologies PicoPure RNA Kit. RNA integrity will be calculated using the Agilent Bioanalyzer 2100 and Illumina s recommended DV200 score. Samples with DV200 < 30% will not be accepted. Whole Blood Samples: If red blood cells are removed from the whole blood sample before RNA isolation, the white blood cell fraction can be extracted using Qiagen RNeasy Mini Kit or equivalent. For whole blood, Qiagen PAXgene Blood RNA Kit or equivalent is recommended. Total RNA Isolated Using a Phenol-Based Method: If you have already prepared total RNA from any biological source material using a phenol-based method, you must clean up the RNA with the Qiagen RNeasy Mini Kit or equivalent. Other Biological Samples: Refer to the existing literature to find isolation protocols for high-quality RNA from other biological samples or contact [email protected]. C. Sending Otogenetics tissues/cells directly: You can submit raw materials to Otogenetics instead of total RNA. Fresh tissues and cell pellets should be flash frozen immediately after harvest or preserved in RNAlater according to the manufacturer s instruction. The samples can be kept in RNA later at room temperature for up to one week; however, shipping is recommended on blue ice. For more information, please contact: [email protected]. IV. RECOMMENDED QUALITY CONTROL A. Determine RNA Concentration and Purity: Use UV spectrophotometric method (e.g. NanoDrop). Measure in RNase-free 10 mm Tris, ph 8.0 buffer. Total RNA concentration should be within limits listed in Table 1. OD260:OD280 ratio should be and OD260:OD230 ratio should be >1.7. B. Assess RNA Integrity: Evaluate RNA integrity using a 1% denaturing RNA gel or an Agilent Technologies 2100 Bioanalyzer (Fig. 1). High quality total RNA samples should give two distinct peaks and yield an RNA Integrity Number (RIN) value greater than Winters Chapel Rd., #100, Atlanta, GA USA [email protected]

3 NOTE: Due to the fragmentation associated with FFPE, the RNA isolated from FFPE samples is always degraded. The integrity of RNA isolated from FFPE samples as determined by the methods described here only serves the purpose of comparing samples, and may help downstream data interpretation. Figure 1. Quality control of total RNA by A) denaturing gel electrophoresis and B) Agilent Bioanalyzer. V. SAMPLE IDENTIFICATION/LABEL RECOMENDATIONS It is important to prevent the sample labels from being dissolved by solvents and the labels from falling off the tubes. Do not use marker pen to mark directly on the tube wall or lid. It is better to write the sample information on a paper/plastic label, stick the label to the tube wall, and secure the label to the tube by wrapping the Scotch tape around the tube for one circle. Please fill out and attach the sample information sheet provided by Otogenetics before shipping the samples. Please make sure that the sample information on the Sample Information Sheet matches the labels on the tubes. Upload the file to LabLink at and print a copy to include with your sample shipment. VI. SAMPLE PACKING RECOMMENDATIONS Please write the sample information on adhesive label and secure the written label to the tube using transparent tape. Include a copy of the Sample Submission Form (provided to you via at time of order) with your sample shipment. Make sure that the sample information on the Sample Submission Form matches the labels on the tubes. For RNA: We recommend 1.5ml or 2 ml screw-cap DNase- and RNase-free microcentrifuge tubes. Please use Parafilm to seal each tube before package. We do not recommend shipping samples dissolved in organic solvents (such as absolute ethanol or isopropanol) because the samples in organic solvents may leak and even cause cross contamination. If it is unavoidable to ship samples in organic solvents, please use screw-cap tubes and seal the opening of the tube with at least 10 layers of Parafilm. RNA samples should be shipped at -20 C using sufficient dry ice for next day delivery. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project Winters Chapel Rd., #100, Atlanta, GA USA [email protected]

4 For snap-frozen cells/tissues: Fresh Tissue Freshly harvested tissues should be flash frozen in liquid nitrogen or an ethanol/dry ice bath and shipped on dry ice immediately. Flash freezing the sample immediately helps to prevent sample degradation. Do not homogenize samples prior to shipment. Tissue samples should never be allowed to defrost. Thawing the sample will cause lipid degradation. Tissues that are not shipped immediately should be stored at 80 C or in liquid nitrogen. Send in 2.0 ml round bottom Eppendorf tubes or corresponding container and cap well. Cotton and absorbent papers can be used to further secure the tubes in the container. RNA samples should be shipped at -20 C using sufficient dry ice for next day delivery. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project. Cell pellet: Remove supernatant from isolated cell pellet. Wash cell pellet to ensure residual media is completely removed; cell pellet should be flash-frozen in liquid nitrogen immediately. Cells that are not shipped should be stored at 80 C. To ensure that you send appropriate quantity, count the cells before freezing. Send cell pellet in 2.0 ml round bottom Eppendorf tubes. Cotton and absorbent papers can be used to further secure the tubes in the container. RNA samples should be shipped at -20 C using sufficient dry ice for next day delivery. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project. For FFPE samples: FFPE samples can be shipped at room temperature. If using microcentrifuge tubes, screw-top lids are preferred. Please use Parafilm to cover lid for added seal precaution and clearly label tubes with water-resistant tags to ensure ink is not removed. Place individual tubes into a 50 ml conical tube filled with packing material (such as paper towels) to absorb the stress of shipping. Fill the void with paper to avoid physical damage during transport. For slides, make sure to properly wrap the slide boxes in bubble wrap and fill the box with packing material to ensure that the boxes are kept in place. During the warmer months (June August), it is advisable to ship the block(s) with a frozen gel ice-pack in order to prevent the melting of paraffin-embedded tissue blocks during transit. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project. For FAC-sorted cells: Should be the same with frozen cell shipment. For double stranded cdna (prior to be made to sequencing library): We recommend 1.5ml or 2 ml screw-cap DNase- and RNase-free microcentrifuge tubes. Please dissolve purified double stranded cdna in TE buffer (ph8.0). Please use Parafilm to seal each tube before package. We do not recommend shipping samples dissolved in organic solvents (such as absolute ethanol or isopropanol) because the samples in organic solvents may leak and even cause cross contamination. If it is unavoidable to ship samples in organic solvents, please use screw-cap tubes and seal the opening of the tube with at least 10 layers of Parafilm Winters Chapel Rd., #100, Atlanta, GA USA [email protected]

5 double stranded cdna samples should be shipped at -20 C using sufficient dry ice for next day delivery. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project. For RNA-Seq libraries (made into sequencing libraries): We recommend 1.5ml or 2 ml screw-cap DNase- and RNase-free microcentrifuge tubes. Please dissolve purified double stranded cdna in EB buffer (Qiagen 19086). Please use Parafilm to seal each tube before package. We do not recommend shipping samples dissolved in organic solvents (such as absolute ethanol or isopropanol) because the samples in organic solvents may leak and even cause cross contamination. If it is unavoidable to ship samples in organic solvents, please use screw-cap tubes and seal the opening of the tube with at least 10 layers of Parafilm. double stranded cdna samples should be shipped at -20 C using sufficient dry ice for next day delivery. A Sample Submission Form (provided by at time of order), a copy of the quote or purchase order, as well as any relevant QC and safety data must accompany each package to be shipped, for each service project. We recommend 1.5ml or 2 ml screw-cap DNase- and RNase-free microcentrifuge tubes. Please use Parafilm to seal each tube before package. We do not recommend shipping samples dissolved in organic solvents (such as absolute ethanol or isopropanol) because the samples in organic solvents may leak and even cause cross contamination. If it is unavoidable to ship samples in organic solvents, please use screw-cap tubes and seal the opening of the tube with at least 10 layers of Parafilm. RNA and solid tissue samples should be kept in dry ice during shipment unless submitted in RNAstable or RNAlater. Blood samples and saliva samples should be shipped at room temperature. VII. COMPLETING THE SAMPLE SUBMISSION MANIFEST The printed Sample Form, completed Billing Form, and a signed Quote Form must be submitted for each sequencing service project. All information on the forms should be filled out. Please submit the completed forms via to [email protected] or fax to before shipping samples to Otogenetics. In addition, please also include a printed copy of each form in the shipment and any QC data for the samples if you have them. VIII. SHIPPING SAMPLES TO OTOGENETICS Disclaimer: The information below constitutes only a recommendation for shipping samples classified as "non-regulated materials" to our facility. Due to continuing changes in regulations, clients should always check with their safety office and/or shipping department to ensure regulatory compliance. To date, RNA and cdna are not defined as diagnostic specimen in the International Air Transport Association (IATA) packing instruction and therefore no special packaging requirements have to be filled Winters Chapel Rd., #100, Atlanta, GA USA [email protected]

6 A. Domestic (US and Canada) Shipping Please arrange for delivery of the shipment weekdays (Mon Fri). Make sure that all samples are prepared and packaged according to the guideline given above. Select reliable couriers. We recommend FedEx ( UPS ( or DHL ( and express next-day service for shipments within the United States, following the federal guidelines for shipping lab specimens. Ship the samples to: Sample Receiving Otogenetics Corporation 4553 Winters Chapel Rd. Suite 100 Atlanta, GA USA the tracking information once the package has left your facility, using Sample Tracking Quote# XXX as the subject for the , including courier name and tracking number to help ensure that the samples arrive safely and without any delay. B. International Shipping Please arrange for delivery of the shipment weekdays (Mon Fri). Make sure that all samples are prepared and packaged according to the guideline given above. Use express services (the fastest delivery offered) from a courier, such as FedEx, DHL, World Courier, UPS or USPS. Contact your local international courier and complete an INVOICE (commercial invoice, customs invoice or pro-forma invoice) which is requested for customs, and include it with the shipment. Please fill the INVOICE as below: RNA or DNA Samples for Research Use Only Non-Dangerous, Non-Infectious No Commercial Value, Value for Customs Only Declare the value of the goods for customs [i.e. $1.00 (USD) or 1.00 (EUR)] Number of samples and volumes [the # of samples, and the estimated volume] Type of container You may be required by the courier to sign a USDA Statement or Toxic Substances control Act Certification (declaration for regulations). Please refer to the information above or check I certify that all the chemical substances in this shipment are not subject to TSCA. Ship the samples to the address provided above. [email protected] the tracking information once the package has left your facility, using Sample Tracking Quote# XXX as the subject for the , including courier name and tracking number to help ensure that the samples arrive safely and without any delay. Priority for sequencing service projects is assigned on a first-come, first-serve basis. Return shipment of leftover DNA or RNA samples following analysis can be arranged for an additional shipping fee. Contact Otogenetics to set up return shipment after receiving your data. Otogenetics will keep RNA and DNA samples under frozen conditions for a maximum of 8 weeks following data release. CLIA Registered #11D State of GA Clinical Laboratory License # Copyright 2015 by Otogenetics Corporation. Products and specifications are subject to review and change without notice Winters Chapel Rd., #100, Atlanta, GA USA [email protected]