Ambient Temperature Stabilization of RNA derived from Jurkat, HeLa and HUVEC Cell Lines for Use in RT-qPCR Assays

Ambient Temperature Stabilization of RNA derived from Jurkat, HeLa and HUVEC Cell Lines for Use in RT-qPCR Assays C. Litterst 1, H. Martinez, B. Iverson and R. Nuňez 1 Bio-Rad Laboratories, Life Science Group, Hercules, CA IntegenX Corporation, Pleasanton, CA Abstract GenTegra RNA is a revolutionary technology for the stabilization, transport and storage of purified RNA. In this study, we report that GenTegra RNA preserves the integrity of purified RNA extracted from Jurkat, HeLa and HUVEC cell lines at ambient temperature during storage in the liquid and dry states, with no effect on performance in gene expression analysis by RT-qPCR. Introduction RNA samples derived from cell lines are frequently used for gene expression analysis in cancer and other research. RNA quality plays a major role in the generation of accurate results from gene expression analysis. Once purified from cells or tissue, RNA stability must be maintained, since RNA is very susceptible to degradation through hydrolysis, especially at elevated temperatures. In addition, RNA is enzymatically degraded by RNases that co-purify with nucleic acids or are introduced from the environment. Preventing degradation of RNA is a major challenge, as is protecting purified RNA during long-term storage and transport. A group of R&D scientists at Bio-Rad Laboratories regularly purify RNA from Jurkat, HeLa and HUVEC cells and are always interested in learning about novel methods of preserving RNA integrity. The use of purified RNA is an important part of the lab s ongoing research to develop new tools for gene expression analysis. RT-qPCR-based gene expression analysis of transcripts such as MAP4 and GAPDH is used as part of the laboratory s work to assess the quality of mrna. To address the difficulties faced by scientists working with purified RNA specimens, IntegenX has developed GenTegra RNA (), a groundbreaking technology that permits long-term storage and transport of purified RNA at ambient temperature in the dry state, while providing an added level of stability for RNA in the liquid state, empowering short-term analysis. is composed of an inert, water-soluble, chemical matrix, which provides protection from oxidation and hydrolysis, conveys thermal stability, and enables quantitative recovery of RNA samples. simplifies sample handling in the liquid state by inactivating trace nucleases, eliminating the need to handle RNA on ice. Here, we demonstrate that storage of purified RNA in has no effect on RNA integrity or performance in RTqPCR, making a safe, convenient method of stabilization, transport and storage of purified RNA. Materials and Methods RNA Purification, Application, Storage and Recovery Total RNA was purified from Jurkat, HeLa and HUVEC cells using the column-based Aurum Total RNA Mini Kit (Bio-Rad, Hercules, CA). The specimens were extracted and labeled as shown in Table 1. Samples were in a volume of -5µl. RNA specimens were applied to tubes immediately after isolation and quantitation. Following application, one set of RNA samples was stored at 5 C or for 4 hr in the liquid state. A second set of RNA samples was dried overnight (1hr) in a FastDryer (IntegenX, Pleasanton, CA) according to the manufacturer s instructions. Samples were then stored in the dry state for 5 days at 5 C, or 5 C. A set of RNA samples was stored under identical conditions with no protection as a control. An 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg 1

Sample - C 5 C 5 C No Protection No Protection Sample - C 5 C 5 C No Protection No Protection aliquot of each RNA sample was stored at - C as a frozen control. Following the dry storage period, the samples were rehydrated in -5µl of molecular grade water according to the manufacturer s instructions. Table 1. Cell line types and mass of DNA applied to each tube. Sample Cell Line Type Mass DNA per Sample 1 Jurkat 1 µg HUVEC 5 µg 3 HeLa 1 µg 4 Jurkat µg RNA Quantitation and Gel Electrophoresis Results Storage of cell line RNA samples with in liquid state RNA aliquots were incubated in the liquid state at 5 C or for 4 hours in the presence and absence of and compared to control samples stored at - C (Table ). Quality of RNA is shown by OD and RQI. There was no difference in RNA integrity for any of the samples stored at 5 C or in the presence of and the frozen controls. On the other hand, samples exhibited significant signs of degradation via RQI following storage at in the absence of GT- RNA. RNA was quantified using a NanoDrop 1 Table. NanoDrop and Experion analysis of RNA samples (ThermoScientific, Wilmington, DE). Information 1-4 stored in the presence or absence of in the liquid state for 4 hr at 5 C or, with controls stored about ribosomal RNA integrity was gathered from an at - C. electrophoretic trace and an RNA Quality Indicator NanoDrop Concentration (ng/µl) (RQI) score was obtained using an RNA Stdsens analysis chip processed by the Experion automated electrophoresis system (Bio-Rad, Hercules,CA) according to the manufacturer s protocol. RQI values are given on a scale of 1 to 1, with 1 being the most degraded RNA profile and 1 being the most intact 1. 1 3 4 45 5 57 cdna Synthesis and Quantitative Real-Time PCR (qpcr) Analysis Synthesis of cdna (µl total) from 1.5µl of total RNA was carried out using the iscript cdna Synthesis Kit according to the manufacturer s protocol (Bio-Rad, Hercules, CA). qpcr was performed on the CFX34 real-time PCR detection system (Bio-Rad, Hercules, 193 3 3 43 445 4 9 77 1 139 59 43 nd nd 75 nd nd Experion RQI Analysis Results CA)..5µl cdna was used with iq Sybr Green 1 1 1 1 9.. Supermix (5µl total reaction) for two genes using 9. 9.9 9. 9.5 1.9 three primer sets according to the manufacturer s 3 1 1 1 9. 1. protocol (Bio-Rad, Hercules, CA). The primer sets 4 1 1 1 9.9 1.9 were specific for the 3 and 5 end of MAP4 (MAP4 3 nd: Not determined due to NanoDrop error and MAP4 5 ) and GAPDH. GAPDH was used as an endogenous control. Electrophoretic patterns for RNA samples stored in the liquid state at 5 C showed no difference between samples stored with or without (data not 19 Corte 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg

shown). However, there was marked degradation in the RNA samples stored at without, as shown by both electrophoretic patterns and RQI scores generated by the Experion (Figures 1 and ), while the samples stored with appeared to be intact and of good quality. - C freezer No Protection 1 3 4 1 3 4 1 3 4 with two different genes, MAP4 and GAPDH (Figure 3). For the MAP4 mrna, primer pairs were chosen that bind to the 5 and 3 ends of the cdna, respectively. As the data shows for GAPDH, there is no degradation noted by RT-PCR for storage at 5 C in RNA samples stored in the liquid state with, indicating that the does not inhibit RT-qPCR. While a small increase in Ct is noted for samples stored in at, a much more significant increase is noted for samples stored with no protection. The other two transcripts for MAP43 and MAP45 show almost identical results (data not shown). had no effect on melt with a small variation in degraded samples (data not shown). Figure 1. Experion eletrophoretic pattern for samples 1-4 stored in the liquid form for 4 hr with and without at. 1. - C 5 C 5 C No No Protection Protection. Sample 1 Sample RQI = 1 RQI = 9.. - C Control 5. 3. RQI = 9. RQI = 9.5 35. sample1 sample sample3 sample4 Figure 3. Ct values of RT-PCR using GAPDH primers and cdna made from RNA samples stored in the liquid state at 5 C and with and without and a control stored at - C. RQI =. RQI = 1.9 No Protection Figure. Experion RQI data for two representative samples for stored in the liquid form at with and without GT- RNA and compared with a control stored at - C. The quality of the mrna was tested using RT-qPCR Storage of cell line RNA samples with in dry state An additional set of RNA aliquots was dried overnight in the presence or absence of and stored at 5 C, or 5 C for 5 days for comparison with a frozen control stored at - C. Storage at and 5 C were used to assess the ability of RNA samples stabilized in the to withstand extreme temperatures that often occur during transport. According to FedEx shipping guidelines, packages can 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg 3

S reach temperatures of approximately C (14 F) for up to one day during transport. At the end of the 5 day storage period, RNA samples were rehydrated in to 5µl of molecular-grade water and quantitated via NanoDrop 1. The values are summarized in Table 3. Quantitative recovery was achieved with samples stored in, regardless of storage condition. Experion RQI values are summarized in Table 4. At 5 C and. there is no statistical difference in RQI in samples stored with. At 5 C, there is some degradation. This is in contrast to the dried RNA samples stored without the. The RQI values for samples stored with no protection are markedly to totally degraded at all temperatures (5 C, and 5 C) with the exception for one outlier at. Table 3. Concentration (ng/µl) of RNA recovered from dried samples stored at 5 C-5 C as compared to control at - C. Sample - C 5 C 5 C No Protection 1 45.3 44.9 5. 19. 17.. 3 43.9 49. 4. 4 747.3 713.9 5.1 Sample - C 1 45.3 47.3 19. 193 3 43.9 411 4 747.3 74. Sample - C 5 C 1 45.3 4.9 19. 194.5 3 43.9 44.5 4 747.3 717.13 3 No Protection 5 57 4.3 491. 7. 5 C No Protection 3 55. 17 1 51.9 39.1 Figure 4 shows Experion electrophoretic patterns corresponding to Table 4 RQI values for two representative RNA samples stored in the dry state for 5 days in the presence or absence of at 5 C. Figures 5 and demonstrate Experion electrophoretic patterns corresponding to RQI values in Table 4 for storing RNA samples in the dry state with and without for 5 days at. Table 4. Experion RQI of dried RNA samples with and without stored at 5 C-5 C, compared to control at - C for 5 days. Sample - C 5 C 5 C No Protection 1 9.3 1..1 9. 9..7 3 1. 5.5*.3 4 1. 9.7 3.4 Sample - C No Protection 1 9.5 9.9 1. 9.7 9. 1.9 3 1..5 7.9 4 1. 9.1 1. Sample - C 5 C 5 C No Protection 1 9.3.3.9 9... 3 1. 7. 3.3 4 1..3 3.1 35 Sample 1 Sample Sam ple 1 Sam ple RQI = 9.3 1 1 5-5 5 3 35 4 45 5 55 5 7 5 Sam ple 5 4 RQI = 1 14 1 1-4 5 3 35 5 3 35 4 45 5 RQI = 9. - C 5 Control 5 3 35 4 45 5 55 5 7 1 Sam ple 1 RQI = 9 4 4 45 5 55 5 7 5 3 35 4 45 5 55 5 7 Sam ple 9 1 Sam ple 1 RQI =.1 55 5 7 14 1 1 4-5 3 35 4 45 5 RQI =.7 55 5 7 No Protection Figure 4. Experion eletropheretic patterns for two representative RNA samples dried with and without GT- RNA for 5 days at 5 C as compared to control in a - C freezer. 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg 4

- C freezer No Protection 1 3 4 1 3 4 1 3 4 35 3 5 1 Sample 1 Sam ple 1 RQI = 9.3 1 14 1 1 4 Sample Sam ple RQI = 9. - C Control 5-5 3 35 4 45 5 5 3 35 4 45 5 55 5 7 55 5 7 Sam ple 5 Sam ple RQI =.3 5 RQI =. 1 5 4 3 1 5 C Figure 5. Gel image of samples 1-4 stored dry in the presence or absence of for 5 days at and a control at - C. 5 3 35 4 45 5 Sam ple 9 1 4 55 5 7 RQI =.9 5 3 35 4 45 5 Sam ple 1 1 1 4 55 5 7 RQI =. 5 C No Protection 11 141 1 4-5 3 35 4 45 5 1 1 1 4 4 Sample Sample 4 Sam ple Sam ple 4 Sam ple 1 5 3 35 4 45 5 Sam ple 5 3 35 4 45 5 RQI = 9.7 55 5 7 RQI = 9. 55 5 7 RQI = 1.9 55 5 7 5 1 5 35 3 5 1 5 45 435 3 5 5 3 35 4 45 5 Sam ple 1 5 3 35 4 45 5 Sam ple -5 5 3 35 4 45 5 RQI = 1 55 5 7 RQI = 9.1 55 5 7 RQI = 1. 55 5 7 - C Control No Protection Figure. Electrophoretic pattern generated by Experion analysis of two representative RNA samples stored in the presence or absence of in the dry state for 5 days at and a control stored at - C. Figure 7 shows the Experion electrophoretic patterns for RNA samples dried in the presence or absence of at 5 C for 5 days. The 5 C temperature is a very harsh condition for RNA. There was some degradation in this sample set in. The RQI were between.3 and.3. In stark contrast, the samples without were totally degraded. 5 3 35 4 45 5 55 5 7-5 3 35 4 45 5 55 5 7 Figure 7. Electrophoretic pattern generated by Experion analysis of RNA samples 1 and, stored in the presence or absence of in the dry state for 5 days at 5 C compared to a control stored at - C. RT-PCR for GAPDH was performed on all dried RNA samples stored with and without at room temperature, and 5 C as well as a control stored at - C in a freezer. See Figure for GAPDH results. RT-PCR was also completed using MAP4(5 ) and MAP4(3 ) primer sets (see Figures 9 and 1). Using all three primer sets, all samples stored with showed no degradation at 5 C or. samples showed some degradation at 5 C for 5 days. The stabilized the RNA at all temperatures. There was <1Ct delay in RT-qPCR samples stored with showing that the RNA samples were of good quality. In contrast, all samples stored without showed marked degradation regardless of temperature while stored for 5 days. 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg 5

1 Control No Protection - C 5 C 5 C 5 C 5 C Control No Protection - C 5 C 5 C 5 C 5 C 17 19 1 3 5 5 7 3 9 35 31 33 4 sample1 sample sample3 sample4 Figure. Ct values of RT-PCR using GAPDH primers and cdna from RNA stored dry at 5 C-5 C with and without and a control stored stored at - C. 35 sample1 sample sample3 sample4 Figure 1. Ct values of RT-PCR using MAP4(5 ) primers and cdna made from RNA stored dry at 5 C, and 5 C with and without and a control at - C. 19 1 Control No Protection - C 5 C 5 C 5 C 5 C The does not significantly affect real-time RT- PCR. In Table 5, the threshold cycles are delineated, and there is no statistical difference between samples that were stored in the presence or absence of GT- RNA. This is graphically represented in Figure 11 by amplification and Figure 1 by melt peaks. 3 5 7 9 31 sample1 sample sample3 sample4 Figure 9. Ct values of RT-PCR using MAP4(3 ) primers and cdna made from RNA stored dry 5 C, and 5 C with and without and a control stored at - C. Table 5. Ct values of GAPDH, MAP4(3 ) and MAP4(5 ) transcripts using samples stored in the presence or absence of. Target Ct GT - RNA Ct - No protection GAPDH 1. 1.7 GAPDH 1.1 1.9 GAPDH 1.13 1. MAP4 (3 ).74.1 MAP4 (3 ).9.5 MAP4 (3 ).71.1 MAP4 (5 ).37.4 MAP4 (5 ).4. MAP4 (5 )..5 19 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg

Figure 11. Amplification represented in PCR cycles and RFUs for samples with and without. Red line: GAPDH; blue line: MAP4(3 ); green line: MAP4(5 ). Figure 1. Melt peak represented in Celsius Temperature and d(rfu)/dt. Red line: GAPDH; blue line: MAP4(3 ); green line: MAP4(5 ). Conclusions In the present study, we examined the use of for storage of total RNA purified from Jurkat, HUVEC, and HeLa cell lines at concentrations varying from 1-µg. is an inert chemical matrix that has the ability to inactivate trace RNase, stabilizing the RNA in the liquid phase for short-term sample handling. Very high quality RNA (RQI of 9.-1) was used for this study. However, even with this exceptional starting material, early signs of RNA degradation were observed in all cell line samples following a 4 hour storage period in the liquid state at in the absence of. These signs of degradation were not observed when the RNA was stabilized with. ensures integrity, stability, and quantitative recovery of purified RNA samples, enabling workflow flexibility. In an ideal scenario, immediately after RNA isolation from cells or tissues, an aliquot destined for prompt use (i.e. for quantitation, gel/experion analysis or any downstream application) is stabilized in and kept in the liquid phase for short-term analysis, while aliquots tagged for long-term storage or transport are dried following stabilization in GT- RNA. In the dry-state, further preserves RNA integrity, protecting RNA samples from hydrolysis, oxidation and exposure to temperatures ranging from - C to 7 C. Here, we have shown that the integrity of RNA stored for 5 days in the dry-state, at 5 C, and 5 C is preserved in the presence of. Conversely, samples stored under the same conditions without GT- RNA exhibited substantial degradation. At 5 C, with, the RQI varied from 9.-1. (except for one outlier sample) with a freezer control of 9.3 to 1.. At 5 C, with no protection, samples had RQI scores of.1-3.4. At, the samples stored in had RQI scores of.5 to 9.9 with a freezer control of 9.5-1.. Samples stored at with no protection exhibited RQIs between 1.9 and 1. Even under the most extreme RNA storage condition of 5 C, samples with exhibited RQIs between.3 and.3 compared to a - C freezer control with RQI of 9.3 to 1.. This is slightly more degraded than samples with stored at 5 C or, but intact RNA is still preserved. On the other hand, samples stored with no protection at 5 C showed RQIs ranging from. to 3.3. The substantial degradation observed in samples stored without at and 5 C demonstrates the ability of to protect against heat-catalyzed hydrolysis and oxidation of the RNA. Storage at and 5 C was used primarily to assess the ability of RNA samples stabilized in the GT- RNA to withstand the extreme temperatures that often occur during transport. According to FedEx shipping guidelines, packages can reach temperatures of approximately C (14 F) for up to one day during 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg 7

the shipping process. Thus, eliminates concerns about sample loss due to high temperature or shipping delays. These results demonstrate that intact RNA can be recovered even after exposure to extreme temperatures for much longer than would be expected during shipping. Finally, we used RNA stored in the presence of GT- RNA for RT-qPCR assays. RNA purified from all cell line types stored in the presence of in liquid form at 5 C and, and in the dry state at 5 C, and 5 C, provided equivalent performance to the frozen controls in qpcr quantification of GAPDH and MAP4 transcripts. Amplification curves and melt peaks demonstrate that the inert chemical matrix of does not inhibit cdna synthesis or performance in qpcr analyses with storage at 5 C or in either the liquid state for 4 hours or in the dry state for 5 days at temperatures of 5 C, or 5 C. Less than one Ct value delay in q-pcr shows the RNA to be of good quality. stabilizes RNA at all temperatures tested and there is no loss of RNA by NanoDrop quantitation. In conclusion, enables storage and transport of purified RNA at ambient temperature. It also provides an added level of stability in the liquid state, enabling short-term sample handling without ice. GT- RNA is compatible with cdna synthesis and qpcrbased expression analysis. Thus, assures preservation of irreplaceable RNA samples without interfering with downstream analyses. References 1. RNA Quality Indicator (RQI) - A New Measure of RNA Integrity. Publication: BioRadiations_117.pdf. Fedex Packaging Pointers: Perishable Shipments.. http://www.fedex.com/us/services/pdf/pkg_ Pointers_Perishable.pdf 57 Stoneridge Drive, Suite 3 Pleasanton, CA 945 www.mygentegra.com www.integenx.com pg