MACSPlex mirna Kit Cancer

Transcription

1 For further information refer to our website For technical questions, please contact your local subsidiary or distributor. Technical Support Team, Germany: Phone MACSPlex mirna Kit Cancer human For up to 96 tests Order no Miltenyi Biotec GmbH Friedrich-Ebert-Straße Bergisch Gladbach Germany Phone Fax Miltenyi Biotec Inc Lindbergh Street Auburn, CA , USA Phone 800 FOR MACS Phone Fax Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use.

2 Content 1. Description Principle of MACSPlex mirna Kit Cancer Applications Reagent and instrument requirements Protocols for assay Sample preparation Fluorescent sample labeling Hybridization assay Flow cytometer setup Setup of the MACSQuant Instrument Setup of other flow cytometers Flow cytometric acquisition and data analysis 21 using the MACSQuant Express Mode 4.1 Flow cytometric acquisition using 21 the MACSQuant Express Modes 4.2 Calculation of relative mirna expression levels Performance Troubleshooting Description Description This product is for research use only. Components 96 MACSPlex mirna Enzyme 1 (lyophilized, 12 red colored 8-tube strips) 96 MACSPlex mirna Enzyme 2 (lyophilized, 12 green colored 8-tube strips) 450 µl MACSPlex mirna Reconstitution Buffer 210 µl DMSO 1 ml Double-distilled Water, RNase-free 400 µl MACSPlex mirna Cancer Beads 100 ml MACSPlex mirna Buffer ml MACSPlex mirna Buffer µl MACSPlex mirna Hybridization Buffer 2 ml MACSPlex mirna Setup Beads 1 2 ml MACSPlex mirna Setup Beads 2 1 MACSPlex Round Bottom Plate 1 MACSPlex Filter Plate 2 adhesive foils 2 3

3 Description Description Size Up to 96 tests Product format MACSPlex mirna Reconstitution Buffer contains DTT and MACSPlex mirna Hybridization Buffer contains tetramethylammonium chloride and sodium dodecyl sulfate. MACSPlex mirna Cancer Beads, MACSPlex mirna Setup Beads 1, and MACSPlex mirna Setup Beads 2 are supplied in buffer containing stabilizer and 0.05% sodium azide. MACSPlex mirna Buffer 1 and MACSPlex mirna Buffer 2 contain 0.05% sodium azide. All buffers included in the kit are evaluated for the absence of RNase activity. Storage Store MACSPlex mirna Cancer Beads, MACSPlex mirna Setup Beads 1, MACSPlex mirna Setup Beads 2, and MACSPlex mirna Reconstitution Buffer protected from light at 2 8 C. Do not freeze. Store MACSPlex mirna Buffer 1 and MACSPlex mirna Buffer 2 at 2 8 C. Store MACSPlex mirna Enzyme 1 and MACSPlex mirna Enzyme 2 at 20 C. Store DMSO, Double-distilled Water, MACSPlex mirna Hybridization Buffer, MACSPlex Round Bottom Plate, MACSPlex Filter Plate, and adhesive foils at room temperature. The expiration dates are indicated on the labels. 1.1 Principle of MACSPlex mirna Kit Cancer MACSPlex mirna Kits are designed for detection of micrornas (mirnas). The analysis is based on MACSPlex (MPx) mirna Cancer Beads, which display defined fluorescence properties and can be identified using standard flow cytometry techniques. Within an array MACSPlex mirna Cancer Beads contain a cocktail of various fluorescently labeled bead populations, each coupled with a specific oligonucleotide complementary to one of the respective mirnas within the sample. The MACSPlex mirna Kits offer a system for semiquantitative analysis of differential mirna expression. After enzymatic labeling of the samples with Vio 670, the samples are incubated with MACSPlex mirna Cancer Beads and the labeled mirnas hybridize to the specific oligonucleotides. The hybridized beads can be analyzed based on the fluorescence characteristics of both the MACSPlex mirna Cancer Beads and the hybridized mirnas. 4 5

4 Description Description MACSPlex mirna Cancer Beads coupled to oligonucleotides Labeled mirnas hybridized to specific MACSPlex mirna Cancer Beads MACSPlex mirna Cancer Bead labeled mirna Figure 1.1: Principle of MACSPlex mirna Kit Cancer. MACSPlex mirna Cancer Beads contain a cocktail of different fluorescently labeled bead populations (as indicated by the different shades of purple above). MACSPlex mirna Cancer Beads consist of 39 bead populations that have been coupled with oligonucleotides specific for the mirnas listed in table 1.1. The 39 bead populations can be distinguished by different fluorescence intensities detected in the B1 and B2 channel of the MACSQuant Analyzer and MACSQuant Analyzer 10 or the FITC and PE channel of other flow cytometers. mirna name Bead population Accession ID mir-378b 22 MIMAT mir-141-3p 23 MIMAT mir-23a-3p 24 MIMAT mir-17-5p 32 MIMAT mir-20a-5p 33 MIMAT mir-200a-3p 34 MIMAT mir-29a-3p 35 MIMAT mir MIMAT mir-143-3p 43 MIMAT mir-21-5p 44 MIMAT mir-19b-3p 45 MIMAT mir-31-5p 46 MIMAT let-7f-5p 52 MIMAT mir-335-5p 53 MIMAT mir-200b-3p 54 MIMAT mir-34a-5p 55 MIMAT

5 Description Description Control-4-Neg 56 n.a. let-7a-5p 57 MIMAT mir-101-3p 63 MIMAT mir-373-3p 64 MIMAT mir-122-5p 65 MIMAT mir MIMAT Control-1-U6 67 n.a. hsa.mir-30b-5p 68 MIMAT mir-15b-5p 74 MIMAT mir-520c-3p 75 MIMAT mir-200c-3p 76 MIMAT Control-2-5.8S 77 n.a. mir-9-5p 78 MIMAT mir-23b-3p 79 MIMAT let-7g-5p 85 MIMAT mir-145-5p 86 MIMAT let-7b-5p 87 MIMAT mir-103a-3p 88 MIMAT mir-10b-5p 89 MIMAT mir-16-5p 96 MIMAT mir-126-3p 97 MIMAT mir-15a-5p 98 MIMAT Control-3-C4 99 n.a. Table 1.1: Bead populations coupled with oligonucleotides specific for the mirnas. MACSPlex-B MACSPlex-B Picture in process Figure 1.2: Detection of MACSPlex mirna Cancer Bead populations in a MACSPlex-B1 (FITC) versus MACSPlex-B2 (PE) dot plot. 8 9

6 Description Description The starting material for detection of mirnas can either be total RNA or size-fractionated total RNA. Depending on the isolation method, small RNAs may get lost. Thus, it is recommend to use total RNA. Total RNA of the samples of interest is labeled with MACSPlex mirna Enzyme 1 and MACSPlex mirna Enzyme 2. After the 2.5-hour labeling procedure the MACSPlex mirna Cancer Beads and the MACSPlex mirna Hybridization Buffer are added to the samples. During a 3-hour incubation period, the mirnas hybridize to the MACSPlex mirna Cancer Beads. The median of the Vio 670 fluorescence (R1/APC channel) of each hybridized bead population is used for the calculation of specific mirna expression levels between samples (see section 4.2). 1.2 Applications The MACSPlex mirna Kit Cancer has been developed for the simultaneous flow cytometric detection of 39 mirnas that are known to be deregulated in different cancer types in a single sample. 1.3 Reagent and instrument requirements Polypropylene or polystyrene reagent tubes for preparation and storage of RNA samples. Vacuum manifold or centrifuge with adapters to accommodate microtiter plates. Orbital thermo shaker for tubes (frequency 1200 rpm) or PCR cycler. PCR cycler or heat block for PCR tubes. MACSQuant Analyzer, MACSQuant Analyzer 10 (# ), or other flow cytometers equipped with blue (488 nm) and red (635 nm) lasers able to discriminate FITC, PE, and APC fluorescence. Note: The MACSQuant VYB cannot be used. MACS Chill 96 Rack (# ), when using the MACSQuant Analyzer or MACSQuant Analyzer 10. MACSQuant Calibration Beads (# ), when using the MACSQuant Analyzer or MACSQuant Analyzer 10. Disposable RNase-free pipette tips RNA to be labeled : Choose a method that preserves small RNAs by using, e.g., mirneasy Mini Kit (50) (Qiagen, # ), TRI Reagent (Sigma-Aldrich, # T9424), or TRIzol Reagent (Invitrogen, # ) for isolation of total RNA. Ice-water bath 10 11

7 Protocols for assay Protocols for assay 2. Protocols for assay Kit components should not be substituted or mixed with those from other kits or lots. Always use RNase-free tubes and pipette tips. Avoid air bubbles Add RNA to MACSPlex mirna Enzyme 1 Incubate 8-tube strips at 37 C Add DMSO, incubate at 90 C, put on ice Add MACSPlex mirna Reconstitution Buffer, add to MACSPlex mirna Enzyme 2 Incubate 8-tube strips at 16 C Add MACSPlex mirna Cancer Beads and MACSPlex mirna Hybridization Buffer Incubate 8-tube strips at 48 C, 1200 rpm Transfer to MACSPlex Filter Plate and wash plate Transfer to MACSPlex Round Bottom Plate Acquire data using the Express Mode of the MACSQuantify Software 2 min 30 min 10 min 5 min 2 hours 2 min 3 hours 10 min 1 min 2.1 Sample preparation RNAs are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when working with the MACSPlex mirna Kit Cancer. In addition, RNase-free reagents, tubes, and barrier pipette tips should be used. For RNA isolation: It is very important to choose a method that preserves small RNAs, for example, mirneasy Mini Kit (50) (Qiagen, # ) as a column-based purification method. The concentration of the RNA should be µg/µl. Alternatively, perform a classical Phenol preparation by using, e.g., TRI Reagent (Sigma-Aldrich, # T9424), or TRIzol Reagent (Invitrogen, # ) followed by an acidic chloroform extraction and isopropanol/ethanol precipitation for isolation of total RNA. Loss of small RNAs is critical during ethanol precipitation. The protocol might have to be adapted to preserve small RNA during the precipitation. As a guideline, it is recommended to add 0.5 volumes isopropanol to 1 volume of the aqueous phase of the chloroform extraction step. After mixing and incubation for 10 minutes at room temperature, centrifuge samples at 12,000 g and 4 C. Carefully aspirate the liquid phase without disturbing the RNA pellet. Wash the pellet using 75% ethanol. It is not necessary to resuspend the pellet as this increases the risk of loosing small RNAs. After washing, centrifuge the samples for 5 minutes at 12,000 g and 4 C. Carefully aspirate the liquid phase and let the pellet air dry. Resupend the RNA using RNase free water and store at 80 C. Figure 2.1: Experimental overview for the assay

8 Protocols for assay Protocols for assay Control the integrity of the total RNA, e.g., by Agilent Bioanalyzer or denaturing gel electrophoresis. As partially degraded RNA might impair the mirna profile, use RNA of good quality, e.g., RIN value above 7 or 28s to 18s RNA ratio of about 2:1. If you use a method with column purification, make sure that the isolated RNA contains the complete fraction of small RNA. Perform the total RNA isolation according to the manufacturer protocol. 2.2 Fluorescent sample labeling Work fast and keep samples protected from light, e.g., cover plate or tubes with aluminum foil, especially during incubation steps. Labeling of 1 μg of total RNA is recommended for most sensitive gene expression analysis. In case less than 1 μg total RNA is available, contact Technical Support for further information (macstec@miltenyibiotec. de). Pipetting of small volumes is error-prone and might lead to artifacts during the normalization. Transferring volumes less than 0.5 μl should be avoided. Before starting Adjust the concentration of the RNA sample to 0.25 µg/µl. If the concentration of the sample is too high, use RNase-free water for dilution. If the volume of RNA is too large, it is recommended to concentrate the RNA sample by using a SpeedVac Concentrator at 45 C. The time length depends on the volume of the sample. Do not prolong SpeedVac incubation after the RNA is dried. 1. Centrifuge or tap down each pellet of MACSPlex mirna Enzyme 1 in the 8-tube strips (red color). Note: Cut the strips, if necessary. 2. Add 4 µl RNA sample to each MACSPlex mirna Enzyme 1 tube. 3. Perform a short spin to collect sample at the bottom. 4. Incubate the reaction at 37 C in a PCR cycler or heat block for 30 minutes. 5. Add 2.1 µl of DMSO to each sample. 6. Incubate at 90 C for 5 minutes in a PCR cycler or heat block. 7. Immediately transfer samples to ice-water bath to snap-cool for at least 2 minutes. 8. Perform a short spin to collect sample at the bottom. 9. Immediately add 3.9 µl of MACSPlex mirna Reconstitution Buffer to each sample to a final volume of 10 µl per reaction mix. Perform a short spin to collect sample at the bottom

9 Protocols for assay Protocols for assay 10. Centrifuge or tap down each pellet of MACSPlex mirna Enzyme 2 in the 8-tube strips (blue color). Note: Cut the strips, if necessary. 11. Immediately resuspend MACSPlex mirna Enzyme 2 by adding 10 µl reaction mixture from step 9 to each pellet. 12. Mix by pipetting up and down and perform a short spin to collect sample at the bottom. 13. Incubate at 16 C for 2 hours in a PCR cycler or thermo block. 14. Proceed to hybridization assay (section 2.3) or store sample at 20 C. 2.3 Hybridization assay Work fast and keep samples protected from light, e.g., cover plate or tubes with aluminum foil, especially during incubation steps. Unknown samples should be run in replicates, for example, in duplicates or triplicates. 1. Incubate labeled sample at 70 C for 2 minutes. 2. Immediately transfer samples to ice-water bath to snap-cool for at least 2 minutes. 3. Perform a short spin to collect sample at the bottom. 4. Resuspend MACSPlex mirna Cancer Beads by vortexing for at least 30 seconds and transfer 4 µl of the beads to each tube containing the labeled samples. Note: When working with more than four samples, vortex the beads briefly after adding them to the fourth sample to prevent settling of the beads. 5. Briefly mix MACSPlex mirna Hybridization Buffer and add 7 µl to each sample. 6. Incubate at 48 C for 3 hours in an orbital thermo shaker at 1200 rpm. Note: If a shaker is not available, put sample in a cycler and vortex the samples every 30 minutes. 7. Immediately proceed to section when using the MACSPlex Filter Plate and to section when using 1.5 ml reagent tubes Protocol for the assay using the MACSPlex Filter Plate Place the MACSPlex Filter Plate on a non-absorbent surface during loading steps and incubation, i.e. remove any tissues from the surface, to prevent the wells from running dry. Cover unused wells of the filter plate for later use with the adhesive foil provided with the kit. Use a multichannel pipettor. Washing steps are described for the use of a vaccum manifold. Alternatively, a centrifuge with an adapter for microtiter plates can be used: Put the MACSPlex Filter Plate on top of a conventional 96-flat bottom microtiter plate without lid and place both into the adapter. Centrifuge at 300 g for 3 minutes at room temperature. 1. Pre-wet required wells of the MACSPlex Filter Plate with 200 μl of MACSPlex mirna Buffer 1 per well and aspirate using a vacuum manifold designed to accommodate the filter plate (max. 300 mbar) until the wells are drained

10 Protocols for assay Protocols for assay 2. Add 160 µl MACSPlex mirna Buffer 1 to each hybridized sample of chapter 2.3, step Transfer samples to the MACSPlex Filter Plate. 4. Apply the filter plate to the vacuum manifold and aspirate until wells are drained. Switch off the vacuum and ventilate the vacuum manifold. 5. Add 200 µl of MACSPlex mirna Buffer 1 to each well and apply vacuum until wells are drained. Switch off the vacuum and ventilate the vacuum manifold. 6. Repeat step Add 200 µl of MACSPlex mirna Buffer 2 to each well and apply vacuum until wells are drained. Switch off the vacuum and ventilate the vacuum manifold. 8. Repeat step Place the filter plate briefly on a tissue to remove residual liquid. 10. Add 85 µl of MACSPlex mirna Buffer 2 to each well, resuspend carefully, and transfer 80 µl of each well to the MACSPlex Round Bottom Plate. Note: Make sure to pipette carefully to avoid penetration of the filter membran and transfer the 80 µl sample volume completely. Fill up to 80 µl with MACSPlex mirna Buffer 2, if necessary. Note: Perform the flow cytometric acquisition on the same day, as prolonged storage of samples can result in increased background and reduced sensitivity. Note: Keep samples protected from light by using the protection lid during the flow cytometric acquisition with the MACSQuant Instrument Protocol for the assay using 1.5 ml reagent tubes Use RNase-free polypropylene or polystyrene tubes 1. Prepare 1.5 ml tubes by pipetting 800 µl MACSPlex mirna Buffer 1 into each tube. 2. Add 160 µl MACSPlex mirna Buffer 1 to each hybridized sample of chapter 2.3, step Transfer samples into prepared 1.5 ml tubes. 4. Vortex the sample for 30 seconds. 5. Centrifuge at 3000 g for 1 minute. 6. Aspirate 900 µl of each supernatant carefully, leaving 80 μl in each tube. 7. Resuspend the MACSPlex mirna Bead pellet in each tube by adding 900 µl of MACSPlex mirna Buffer 2 and pipetting up and down. 8. Vortex the sample for 30 seconds. 9. Centrifuge at 3000 g for 1 minute. 10. Aspirate 900 µl of each supernatant carefully, leaving 80 μl in each tube. 11. Transfer 80 µl sample of each tube to the MACSPlex Round Bottom Plate. Note: Make sure to transfer 80 µl sample volume to the round bottom plate. Fill up to 80 µl with MACSPlex mirna Buffer 2, if necessary. Note: Perform the flow cytometric acquisition on the same day, as prolonged storage of samples can result in increased background and reduced sensitivity. Note: Keep samples protected from light by using the protection lid during the flow cytometric acquisition with the MACSQuant Instrument

11 Flow cytometer setup 3. Flow cytometer setup The kit includes MACSPlex mirna Setup Beads 1 and MACSPlex mirna Setup Beads 2 for flow cytometer setup. MACSPlex Setup Beads are not required when using the MACSQuant Analyzer or the MACSQuant Analyzer 10. Note: The MACSPlex mirna Setup 2 can be used to control proper recognition of all bead populations. This kit is not suitable for use with the MACSQuant VYB. 3.1 Setup of the MACSQuant Instrument Calibrate the MACSQuant Instrument using MACSQuant Calibration Beads (# ). For details, refer to the data sheet of the MACSQuant Calibration Beads. After successfully finishing the calibration, the MACSQuant Instrument is ready for measurement. No further setup is required. All necessary setup steps are performed automatically during calibration. 3.2 Setup of other flow cytometers The analysis of MACSPlex mirna Kit Cancer requires flow cytometers with blue (e.g. 488 nm) and red (e.g. 635 nm) lasers, which are capable of detecting FITC, PE, and APC. For setting up these instruments, the MACSPlex Setup Beads 1 and 2 are included in the kit. For instructions on the setup procedures of other flow cytometers, please refer to the application note "General instructions for MACSPlex mirna Kit Cancer" available at Flow cytometric acquisition and data analysis using the MACSQuant Express Mode 4. Flow cytometric acquisition and data analysis using the MACSQuant Express Mode The analysis of the hybridized samples is performed in two steps. First, the samples are automatically measured and the different bead populations are automatically gated by using the appropriate Express Modes of the MACSQuantify Software (see section 4.1). Second, the primary data generated by the Express Mode is used for further calculations and relative quantification of mirna expression levels (see section 4.2). 4.1 Flow cytometric acquisition using the MACSQuant Express Mode To perform the acquisition and data analysis of the MACSPlex mirna Kit Cancer with the MACSQuant Instrument it is highly recommended to use the Express Mode MACSPlex_miRNA to achieve automated measurement and generation of primary data. For details refer to the special protocol Data acquisition and analysis of MACSPlex mirna Kits using the MACSQuant Analyzer Express Modes available at The minimum version number of the Express Mode package needed to run the assay on the MACSQuant Instrument can be found there as well. Note: It is recommended to use the MACSPlex mirna Setup 2 to get familiar with the Express Mode and to control proper recognition of all bead populations. Instead of an mirna sample, 200 µl MACSPlex mirna Setup Beads 2 are used for analysis. 4.2 Calculation of relative mirna expression levels The Express Mode analysis results in a table listing the median signal intensity for all mirnas. The data analysis consists of the following steps: Background subtraction 20 21

12 Flow cytometric acquisition and data analysis using the MACSQuant Express Mode Performance Calculation of normalization factor Normalization of detected signals Determination of relative mirna expression levels 1. Substract the median signal intensity obtained from the background control Control-4-Neg from all other signal intensities of the respective sample. 2. Repeat step 1 for all samples to be analyzed. 3. Select the normalization control Control-3-C4 of all samples, choose the lowest Control-3-C4 signal intensity of all samples and divide all Control-3-C4 signal intensities by this lowest signal intensity. Thus a normalization factor is obtained for each sample. 4. Divide the signal intensities of all mirnas of each sample by the normalization factor of the respective sample. 5. (Optional) If you performed replicates, calculate the mean signal intensity for each mirna of the replicates. 6. Determine the relative mirna expression level by dividing the signal intensities of the samples of interest. Depending on the sample the Control-1-U6 and Control-2-5.8S might also be suitable for normalization instead of the spiked-in control Control-3-C4. For evaluation of the mirnas expression results, it is recommended to consider only mirnas that are expressed two standard deviations above background (limit of detection) in at least one of the two samples that are compared. The limit of detection is two standard deviations above the mean of median signal intensity of all mirnas of the negative control (0 µg RNA). Normalization procedure with Control-3-C4 does not normalize for RNA quality or input material. 5. Performance The assay sensitivity, specificity, and reproducibility of the MACSPlex mirna Kit Cancer was tested by hybridizing RNA of breast cancer tumor samples, cell lines, and synthetic RNA. The detection limit depends on the melting temperature of the respecitive mirna and has been detected to be as low as 0.1 fmol. However, we recommend to use 1 fmol synthetic RNA for reliable experiments with mirna standards. The specificity was among others investigated by using closely related mirnas such as the let-7 family members. In general, mirnas differing in at least one single nucleotide can be distinguished. The mean coefficient of variation of mirna signals detected on replicate samples is usually around 8%. The regression coefficient of expression profiles detected for samples hybridized independently on different beadarrays is approximately

13 Troubleshooting Troubleshooting 6. Troubleshooting The following section offers solutions for problems that might be encountered when using MACSPlex mirna Assays. Variation between replicate samples: MACSPlex mirna Cancer Beads can settle down. Vortex the MACSPlex mirna Cancer Beads briefly at the latest after pipetting of four samples. Low bead number in samples: Mix MACSPlex mirna Cancer Beads sufficiently before pipetting. Ensure that the instrument is calibrated for the relevant 96-well plate to avoid aspiration of air. Avoid aspiration of beads during washing steps. Do not wash or resuspend beads in volumes higher than recommended. High background: Avoid cross-well contamination. Ensure pipetting with multichannel pipets and avoid touching reagent in the plate. The sample may be too concentrated. Test various sample dilutions. Check RNA quality. High background implies that impurities, such as cell debris including proteins, carbohydrates, or salts, are binding to the probe on the beadarray in a non-specific manner. If these substances show fluorescence signals at the scanning wavelength 635 nm, they give rise to background. Make sure to use the washing conditions as described in the protocol. Little or no detection of mirna in sample: Although the MACSPlex mirna Kit Cancer Reagents are tested to be RNase-free, RNAs are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when doing RNA preparation and when working with the kit. In addition, RNase-free reagents, tubes, and barrier pipette tips should be used. The MACSPlex mirna Cancer Kit Reagents are tested for labeling efficiency and stability. However, wrong storage conditions of the reagents or inappropriate labeling conditions might impair the labeling reaction. The lyophilized labeling enzyme might attract water if not stored dry. The lyophilized enzyme should always be visible as white pellet. If the pellet appears translucent or not visible, the enzyme has probably been dissolved and lost its activity. The correct temperature during the labeling reaction is very important. Higher temperatures can lead to insufficient labeling of the RNA. Incorrect incubation temperatures during hybridization or washing can lead to no or weak signals. Fluorescent dyes are susceptible to photobleaching. Avoid prolonged exposure of the fluorescent sample to direct light. Sample may be too dilute. Test various sample dilutions. Use positive and negative control samples. Check the median signal intensity of Control-3-C4 that could serve as internal positive control. Beads not in region or gate: Ensure proper calibration of the MACSQuant Instrument. Samples containing organic solvents or samples of high viscosity should be diluted or dialyzed, respectively

14 Troubleshooting Signal for whole plate is same as background. Control-3-C4 shows no signal: Incorrect amount or no MACSPlex mirna Reconstitution Buffer was added. High variation in samples: Pipette may not be calibrated. Washing was not uniform. Samples may have contained high particulate matter or other interfering substances. Plate agitation was insufficient. Cross-well contamination. Change pipette tips for each well when touching the reagent. Filter plate will not vacuum: Vacuum pressure is insufficient. Increase vacuum pressure. Plate leakage: Vacuum pressure is too high. Adjust vacuum pressure to maximal 300 mbar. Place the MACSPlex Filter Plate on a non-absorbent surface during filling steps and incubation, i.e., remove any tissues from the surface, to prevent the wells from running dry. Avoid touching the plate filter with the tip of the pipette when adding reagents to the wells. Warnings Reagents contain sodium azide. Under acidic conditions sodium azide yields hydrazoic acid, which is extremely toxic. Azide compounds should be diluted with running water before discarding. These precautions are recommended to avoid deposits in plumbing where explosive conditions may develop. Warranty The products sold hereunder are warranted only to be free from defects in workmanship and material at the time of delivery to the customer. Miltenyi Biotec GmbH makes no warranty or representation, either expressed or implied, with respect to the fitness of a product for a particular purpose. There are no warranties, expressed or implied, which extend beyond the technical specifications of the products. Miltenyi Biotec GmbH s liability is limited to either replacement of the products or refund of the purchase price. Miltenyi Biotec GmbH is not liable for any property damage, personal injury or economic loss caused by the product. MACS, MACSQuant, and Vio are registered trademarks and MACSQuantify is a trademark of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for diagnostic or therapeutic use. Copyright 2016 Miltenyi Biotec GmbH. All rights reserved