TaqMan Low Density Array: Performance Parameters Across...
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Kathy Y Lee, Glo-Stella Concepcion, Kelly Li, Jake Freudenthal, Kathleen Shelton, Nico Tuason; Applied Biosystems, Foster City, CA 94404 TaqMan ® Low Density Array: Performance Parameters Across Arrays and Human Endogenous Control Panel Application ABSTRACT The TaqMan ® Low Density Array allows for rapid screening of many samples with 10s to100s of TaqMan ® Gene Expression Assays. The researcher can easily move from gene lists and single assays in tubes to large scale real-time PCR gene expression profiling without the the need for liquid handling robots. Of particular concern to customers is assay performance on the Low Density Array relative to plates, as well as the precision within arrays, across arrays and between manufactured lots. Over the past two years we have accumulated a large amount of data on TaqMan ® Array performance via our QC process and we used this to evaluate its performance. In these studies we show the performance parameters of TaqMan Arrays and the results of 32 tissues run on the TaqMan ® Low Density Human Endogenous Control Array. INTRODUCTION The TaqMan Low Density Array, designed to be used with the Applied Biosystems 7900HT Fast Real-Time PCR System, is a consumable consisting of 384 wells connected by a series of microfluidic channels. During the manufacturing process selected TaqMan Gene Expression Assays are loaded and dried in the array wells as specified by the customer or as defined by a fixed Array Gene Panel (Immune Profiling Array or Endogenous Control Panel). Each manufactured lot is tested for reproducibility by running Universal RNA across all wells; replicates must meet certain criteria before the arrays are released to the customer. To compare performance between plate and low density array, we looked at QC data generated from > 3,000 TaqMan ® Assays that were run in parallel on TaqMan Low Density Arrays and 384-well plates. These data show that assays on the array can discriminate 2-fold change with similar sensitivity as on plates. We used data from multiple manufactured lots of the Immune Profiling Array to determine assay reproducibility and found that across arrays reproducibility was very good; across manufacturing lots the standard deviation increased but was well within the QC standards set for within array reproducibility As a demonstration of the TaqMan ® Low Density Human Endogenous Control Array (P/N 4347601) performance, we screened 32 different tissues and show the variation of expression of 16 common housekeeping genes across these tissues. METHODS • Arrays from 6 different manufacturing lots of the Immune Profiling Array (Fig. 2) were used to test assay performance. cDNA was prepared from commercially available total RNA (Stratagene), using Applied Biosystems High Capacity cDNA Archive Kit for reverse transcription. cDNA was mixed with Universal Master Mix and loaded into the fill ports, 100ng cDNA/port (Fig. 1). The Array was then centrifuged to load the sample specific reaction mixes into the individual wells of the microfluidic card, and sealed before loading into the Applied Biosystems 7900HT System. C T (cycle threshold) values were used to measure reproducibility (4 replicates/gene/sample/card). • RT-PCR was performed on total RNA samples from 32 different tissues (Ambion) and loaded into fill ports on 4 TaqMan ® Human Endogenous Control arrays (Fig. 8). The average C T (3 replicates/gene/sample) was used to calculate the average C T and StDev across the 32 tissues. Figure 1. Figure 2. TaqMan ® Low Density Immune Profiling Array Running The Low Density Array Seal Load Spin Run Real-Time PCR Figure 3. Uniformity across an Array 384 wells containing TGFB1C T Stdev of CT = 0.097 Figure 4a. GAPD (Hs99999905_m1) 8 replicates across 4 cards Figure 4b. Reproducibility Across Arrays 10 15 20 25 30 35 GAPDH IL-8 MCP-1 Rantes Bax PGK1 CD86 IL-18 Ct Card1 Card2 Card3 Card4 Figure 5. Reproducibility of Individual Wells Across Lots Figure 6. Reproducibility Across Lots Immune Profiling Card R 2 = 0.9708 0 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 CT wells Lot_1 CT wells Lot_2 0 5 10 15 20 25 30 35 GAPD IL8 MCP-1 Rantes BAX PGK1 CD86 IL18 Assay CT Lot 1 Lot 2 Lot 3 Lot 4 Figure 7. Ability of TaqMan Low Density Arrays to Discriminate Fold Change in Gene Expression 3 C T TaqMan Low Density Arrays and 384-well plates for different threshold cycles (C T ) is shown below.The results are based on a statistical model of precision (standard deviation) derived from measurement of thousands of TaqMan Assays across hundreds of each consumable. The expression level measured as C T value is shown on the X-axis; the Y-axis shows the fold-change in expression that is measured with 95% confidence at a given C T . Figure 8. TaqMan ® Low Density Human Endogenous Control Array Col 1- 3 Col 4 - 6 Col 7 - 9 Col 10 - 12 Col 13 - 15 Col 16 - 18 Col 19 - 21 Col 21 - 22 A 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 B PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ C 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 D PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ E 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 F PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ G 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 H PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ I 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 J PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ K 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 L PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ M 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 N PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ O 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8 P PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ Figure 9. C T values for 32 tissues, 4 Candidate Endogenous Controls 0 5 10 15 20 25 30 18S GAPDH HPRT RPLPO CT UHR Brain Liver Lung Testis Pancreas Placenta Thymus Thyroid Salivary Gland Mammary Gland Colon Heart Kidney Skeletal Muscle Prostate Spleen Spinal Cord Fetal Kidney Fetal Thymus Fetal Liver Fetal Brain Adrenal Gland Bone Marrow Retina PBL* Tonsil Trachea Uterus Small Intestine Skin Table 1. C T and StDev for 32 tissues with16 candidate endogenous control genes Gene ID CT StDev Gene ID CT StDev RPLPO 20.48 0.68 POLR2A 23.85 1.07 18S 7.06 0.72 TBP 27.00 1.15 HMBS 26.43 0.72 ACTB 19.47 1.27 UBC 20.94 0.79 B2M 19.74 1.27 PPIA 21.45 0.94 HPRT 25.55 1.29 PGK1 23.01 0.96 TFRC 25.38 1.35 IPO8 26.44 1.00 GAPDH 19.77 1.42 GUSB 24.09 1.04 YWHAZ 26.92 1.44 Conclusions • Gene expression measurements on TaqMan Low Density Arrays are highly reproducible, both within and across arrays (Figs. 3, 4, 5, 6). • The high precision of the TaqMan Low Density Array allows discrimination of relative gene expression changes of two-fold or greater (Fig. 7). •The Human Endogenous Control Array can be used to quickly screen a large number of samples to identify candidate normalization genes (Figs. 8, 9; Table 1). For Research Use Only. Not for use in diagnostic procedures. The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. Applied Biosystems is a registered trademark and AB (Design) and Applera are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.TaqMan is a registered trademark of Roche Molecular Systems, Inc. Micro Fluidic Card developed in collaboration with 3M Company. © 2005 Applied Biosystems. All rights reserved. 127PR15-01
Transcript of TaqMan Low Density Array: Performance Parameters Across...
Kathy Y Lee, Glo-Stella Concepcion, Kelly Li, Jake Freudenthal, Kathleen Shelton, Nico Tuason; Applied Biosystems, Foster City, CA 94404
TaqMan® Low Density Array: Performance Parameters Across Arrays and Human Endogenous Control Panel Application
ABSTRACTThe TaqMan® Low Density Array allows for rapid screening of many samples with 10s to100s of TaqMan® Gene Expression Assays. The researcher can easily move from gene lists and single assays in tubes to large scale real-time PCR gene expression profiling without the the need for liquid handling robots. Of particular concern to customers is assay performance on the Low Density Array relative to plates, as well as the precision within arrays, across arrays and between manufactured lots. Over the past two years we have accumulated a large amount of data on TaqMan® Array performance via our QC process and we used this to evaluate its performance. In these studies we show the performance parameters of TaqMan Arrays and the results of 32 tissues run on the TaqMan® Low Density Human Endogenous Control Array.
INTRODUCTIONThe TaqMan Low Density Array, designed to be used with the Applied Biosystems 7900HT Fast Real-Time PCR System, is a consumable consisting of 384 wells connected by a series of microfluidic channels. During the manufacturing process selected TaqMan Gene Expression Assays are loaded and dried in the array wells as specified by the customer or as defined by a fixed Array Gene Panel (Immune Profiling Array or Endogenous Control Panel). Each manufactured lot is tested for reproducibility by running Universal RNA across all wells; replicates must meet certain criteria before the arrays are released to the customer. To compare performance between plate and low density array, we looked at QC data generated from > 3,000 TaqMan® Assays that were run in parallel on TaqMan Low Density Arrays and 384-well plates. These data show that assays on the array can discriminate 2-fold change with similar sensitivity as on plates. We used data from multiple manufactured lots of the Immune Profiling Array to determine assay reproducibility and found that across arrays reproducibility was very good; across manufacturing lots the standard deviation increased but was well within the QC standards set for within array reproducibility As a demonstration of the TaqMan®
Low Density Human Endogenous Control Array (P/N 4347601) performance, we screened 32 different tissues and show the variation of expression of 16 common housekeeping genes across these tissues.
METHODS• Arrays from 6 different manufacturing lots of the Immune Profiling Array (Fig. 2) were used to test assay performance. cDNA was prepared from commercially available total RNA (Stratagene), using Applied Biosystems High Capacity cDNA Archive Kit for reverse transcription. cDNA was mixed with Universal Master Mix and loaded into the fill ports, 100ng cDNA/port (Fig. 1). The Array was then centrifuged to load the sample specific reaction mixes into the individual wells of the microfluidic card, and sealed before loading into the Applied Biosystems 7900HT System. CT (cycle threshold) values were used to measure reproducibility (4 replicates/gene/sample/card). • RT-PCR was performed on total RNA samples from 32 different tissues (Ambion) and loaded into fill ports on 4 TaqMan® Human Endogenous Control arrays (Fig. 8). The average CT (3 replicates/gene/sample) was used to calculate the average CT and StDevacross the 32 tissues.
Figure 1.
Figure 2. TaqMan® Low Density Immune Profiling Array
Running The Low Density Array
SealLoad Spin Run Real-Time PCR
Figure 3. Uniformity across an Array 384 wells containing TGFB1CT Stdev
of CT = 0.097
Figure 4a. GAPD (Hs99999905_m1)8 replicates across 4 cards
Figure 4b. Reproducibility Across Arrays
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Figure 5. Reproducibility of Individual Wells Across Lots
Figure 6. Reproducibility Across Lots Immune Profiling Card
R2 = 0.9708
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Figure 7. Ability of TaqMan Low Density Arrays to Discriminate Fold Change in Gene Expression
3 CT
TaqMan Low Density Arrays and 384-well plates for different threshold cycles (CT) is shown below.The results are based on a statistical model of precision (standard deviation) derived from measurement of thousands of TaqMan Assays across hundreds of each consumable. The expression level measured as CTvalue is shown on the X-axis; the Y-axis shows the fold-change in expression that is measured with 95% confidence at a given CT.
Figure 8. TaqMan® Low Density Human Endogenous Control ArrayCol 1- 3 Col 4 - 6 Col 7 - 9 Col 10 - 12 Col 13 - 15 Col 16 - 18 Col 19 - 21 Col 21 - 22
A 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8B PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZC 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8D PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZE 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8F PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZG 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8H PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZI 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8J PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZK 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8L PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZM 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8N PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZO 18S ACTB B2M GAPD GUSB HMBS HPRT1 IPO8P PGK1 POLR2A PPIA RPLPO TBP TFRC UBC YWHAZ
Figure 9. CT values for 32 tissues, 4 Candidate Endogenous Controls
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UHRBrainLiverLungTestisPancreasPlacentaThymusThyroidSalivary GlandMammary GlandColonHeartKidneySkeletal MuscleProstateSpleenSpinal CordFetal KidneyFetal ThymusFetal Liver Fetal BrainAdrenal GlandBone MarrowRetinaPBL*TonsilTracheaUterusSmall IntestineSkin
Table 1. CT and StDev for 32 tissues with16 candidate endogenous control genes
Gene ID CT StDev Gene ID CT StDevRPLPO 20.48 0.68 POLR2A 23.85 1.07
18S 7.06 0.72 TBP 27.00 1.15HMBS 26.43 0.72 ACTB 19.47 1.27UBC 20.94 0.79 B2M 19.74 1.27PPIA 21.45 0.94 HPRT 25.55 1.29PGK1 23.01 0.96 TFRC 25.38 1.35IPO8 26.44 1.00 GAPDH 19.77 1.42
GUSB 24.09 1.04 YWHAZ 26.92 1.44
Conclusions• Gene expression measurements on TaqMan Low Density Arrays are highly reproducible, both within and across arrays (Figs. 3, 4, 5, 6).
• The high precision of the TaqMan Low Density Array allows discrimination of relative gene expression changes of two-fold or greater (Fig. 7).
•The Human Endogenous Control Array can be used to quickly screen a large number of samples to identify candidate normalization genes (Figs. 8, 9; Table 1). For Research Use Only. Not for use in diagnostic procedures.The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. Applied Biosystems is a registered trademark and AB (Design) and Applera are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.TaqMan is a registered trademark of Roche Molecular Systems, Inc. Micro Fluidic Card developed in collaboration with 3M Company. © 2005 Applied Biosystems. All rights reserved.
127PR15-01
