Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.
We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.
Vascular endothelial cells form the inner cellular lining of blood vessels in the closed cardiovascular system of higher species. They form the interface between the blood and tissues and are characterized by luminal and abluminal surfaces. The endothelium is a diverse, active, and adaptive system that regulates blood flow, nutrient trafficking, immunity, and the growth of new blood vessels1. In the body, endothelial cells normally exist in an environment where they are exposed to the frictional force of circulation, shear stress2. Shear stress is an important regulator of endothelial cell gene expression3, and endothelial cells attempt to maintain shear stress within a given range2,4. Endothelial cells demonstrate angiogenic patterning in the absence of shear stress5 that can improve tissue perfusion. Regional patterns of disturbed flow and altered shear stress are associated with the expression of inflammatory genes6 and the development of atherosclerosis7,8. Thus, models that include shear stress are a major component of understanding endothelial gene regulation.
We describe a method for studying the gene regulation in vascular endothelial cells under shear stress. This system uses non-pulsatile flow and mimics fluid shear stress levels and oxygen concentration that model conditions for arterial endothelial cells. This protocol includes details of methods for the gene knockdown using RNA interference (RNAi), the set-up for the application of shear stress using the parallel-plate flow apparatus, and methods for the spike-in of an exogenous reference RNA prior to analysis by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This pipeline is used for studying gene regulation in endothelial cells in the presence and absence of laminar shear stress and includes an adaptation of the parallel-plate flow apparatus described by Lane et al.9. This particular set-up was designed to facilitate the simultaneous assessment of multiple experimental conditions that allows direct comparison of shear stress conditions, as well as the normalization of RNA analysis. A large heated unit with controlled humidity is utilized to allow multiple separate flow chambers and pumps to be running simultaneously with flow rates monitored for each flow chamber assembly in real-time. The application of this set-up is used for gene knockdown using RNAi in the setting of laminar flow/shear stress, but aspects of this protocol can be applied to any assessment of RNA expression.
Common approaches to the application of shear stress for endothelial cells include microfluidic systems10, a cone-and-plate viscometer11, and a parallel-plate flow chamber12. Microfluidic systems from various manufacturers have been useful in studying mechanobiology and mechanotransduction in multiple cell and tissue types and a variety of biophysical stimuli. For endothelial cells, they have been used to study endothelial cells in isolation, as well as the interaction of endothelial cells and the trafficking of immune or tumor cells10. However, these systems are less suitable for the recovery of large numbers of cells9. Both the cone-and-plate viscometer and parallel-plate flow chambers allow the recovery of large numbers of cells in confluent monolayers12. These systems can generate a range of shear forces and patterns12. The parallel-plate flow chamber assembly9 has the advantage that real-time imaging can be performed through the glass window to evaluate cellular morphology at any time point. Furthermore, the perfusate can be collected under sterile conditions. For the system presented here, the flow can also be monitored in real-time and in a multi-chamber set-up, which facilitates the maintenance of shear conditions between chambers.
For representative experiments, human umbilical vein endothelial cells (HUVEC), which represent a macrovascular endothelial cell type, are used, and the shear stress conditions we use (1 Pa) reflect arterial conditions (0.1 – 0.7 Pa). However, this protocol can be used with other endothelial cell types, and the shear stress conditions can be adjusted according to the experimental question. For example, the evaluation of human endothelial cells in conditions that model venous circulation would require lower levels of shear stress (1 – 6 Pa) and studies that model microvascular circulation have utilized shear stress levels of 0.4 – 1.2 Pa13,14. In addition, shear stress can vary even between endothelial cells within the same blood vessel6. In the current set-up, a single monitoring system is used that can simultaneously monitor four separate flow loops. For labs that need more flow loops, there is space in the dedicated environment for an additional monitoring system.
RT-qPCR is used for the absolute quantitation of gene expression in the setting of shear stress. The relative expression of target genes is often used to compare RNA expression across conditions. Some RNA species can exist at very low quantities or be absent, thus complicating relative measurements. For example, long noncoding RNAs in endothelial cells can exert potent effects at relatively low copy numbers per cell5. In addition, differences in primer efficiency can lead to an inaccurate interpretation from utilizing the delta-delta cycle threshold (Ct) method to analyze the data. To address this concern, we perform absolute quantitation by generating a standard curve using a known quantity of plasmid DNA. Furthermore, complementary DNA (cDNA) synthesis is an inefficient process, and differences in cDNA efficiency can account for differences in RNA expression between conditions and between samples15. The application of shear stress and/or transfection reagents can affect cell proliferation, apoptosis, and viability, or add components that may interfere with RNA isolation and/or cDNA synthesis. To account for the possibility of bias from RNA isolation and cDNA synthesis, we use a spike-in RNA control synthesized in the lab, added at the time of RNA extraction and measured with each cDNA synthesis via RT-qPCR. This allows not only the adjustment for technical differences in RNA extraction and cDNA synthesis but also allows the calculation of absolute quantities per cell, when the cell count is known.
This system uses additional steps to maintain similarity or account for technical differences between conditions. We particularly emphasize these steps because of the complex nature of these experiments, which involve multiple physical set-ups and experimental conditions that can lead to experimental variability.
1. Preparation of Exogenous Reference RNA
NOTE: Choose an exogenous reference RNA that does not exist in the species or model of interest. For mammalian systems, firefly luciferase RNA may be used.
2. Slide Coating
NOTE: Steps 2.1 – 2.10 should be performed 24 – 48 h prior to the anticipated cell seeding.
3. Cell Seeding onto Glass Slides
4. Small Interfering RNA (siRNA) Transfection
5. Calculation of the Flow Rate Based on the Desired Shear Stress9
6. Set-up of a Dedicated Environment for Monitoring System and Multiple Parallel-plate Flow Chambers (Figure 2)
7. Set-up of the Parallel-plate Flow Apparatus
NOTE: For the manufacturing of parallel plates, please see Lane et al.9.
8. Harvesting of the Cells and Extraction of RNA from the Flow Chamber
9. Calculation of the Efficiency of the RNA Extraction and cDNA Synthesis
NOTE: Calculate the luciferase efficiency after RT-qPCR by comparing the theoretical yield and the experimental yield.
Successful linearization of luciferase plasmid using restriction enzymes was confirmed by running digested products on an agarose gel (Figure 1). The size of the linearized product was confirmed using DNA ladders and by comparison with uncut plasmid.
We have adapted the parallel-plate flow chamber set-up from Lane et al.9 for experiments that require multiple conditions/treatments with shear stress or multiple shear stress conditions. We use a dedicated environment, the BEACH, that can house multiple, fully-assembled flow circuits that all have monitored flow rates (Figure 2). The flow rate is monitored just upstream of the parallel-plate assembly (Figure 3). The flow circuits and rates can be monitored directly through glass doors without causing fluctuations in temperature, humidity, or gas content within the BEACH.
Manufacturing processes can lead to small variations in chamber height. Thus, flow rates must be calculated for each chamber to achieve the same shear stress (Table 1). In theory, chambers with identical heights can use identical flow rates to achieve the same shear stress and can be used in series. Typical experiments with endothelial cells use shear stress of 0 – 1.5 Pa. Laminar shear stress of 1 Pa was used in this workflow to model arterial endothelial shear stress. There can also be variations between pump head settings and within pump heads over time with use. Using the flow meter can account for these differences.
Experiments using the application of shear stress often involve multiple shear stress conditions, treatment conditions, and time points. Where possible, we use an endogenous reference RNA to account for any variabilities in the experimental set-up. For some experiments, finding an endogenous reference RNA with quantitative stability is not feasible16. Furthermore, quantitative stability or instability of endogenous reference genes between samples can be attributed to either stimulus-dependent effects on cellular expression levels or variations in efficiencies of RNA extractions or reverse transcription. To account for these inefficiencies, and in the setting where an endogenous reference gene is not quantitatively stable, we use a spike-in exogenous reference gene. For experiments incorporating laminar shear stress in mammalian endothelial cells, we use a firefly luciferase RNA as an exogenous RNA spike-in (Figure 4A).
Figure 4 shows analyzed RT-qPCR experimental data from shear stress experiments assessing Krüppel-like factor 2 (KLF2) loss-of-function using siRNA. KLF2 is a transcription factor upregulated by laminar flow in endothelial cells and a major transcriptional mediator of endothelial gene expression in the setting of laminar flow2.
Figure 4A shows luciferase efficiencies for three separate experiments, each using two flow chambers. Luciferase efficiencies can be similar between samples of an experiment (Experiment 1) or show some variability (Experiments 2 and 3) (Figure 4A). These results are especially valuable in experimental systems where only small absolute changes are seen. The use of an exogenous reference gene may be particularly important in experiments where experimental treatments can interfere with the efficiency of reverse transcription or PCR17. The results depicted in Figure 4A are typical. A luciferase efficiency of 5% indicates that 5% of the luciferase RNA (i.e., the initial starting amount of RNA) added to the sample prior to RNA extraction is detected by RT-qPCR. Between samples or conditions within a single experiment, luciferase efficiencies are usually ± 50%. Results should be interpreted with caution if the variability of luciferase efficiencies is > 50% and should include a review of the experimental procedures and conditions.
Figure 4B shows typical experimental results of RT-qPCR from repeated shear stress experiments, each using multiple parallel-plate flow chambers. Within each experiment, KLF2 mRNA expression is normalized in three ways. The first normalization uses an endogenous reference RNA, Cyclophilin A (CycA). The second normalization uses an exogenous reference RNA, firefly luciferase (Luc). The third normalization uses both the endogenous and exogenous reference RNA. Within each experiment shown in Figure 4B, all three normalization methods (normalization to the endogenous reference gene, the exogenous reference gene, and both endogenous and exogenous reference genes together) yields similar results. If the normalization method significantly changes the results (e.g., leads to > 50% difference), the results should be interpreted with caution. When there is considerable variability between the methods of normalization, the endogenous reference gene(s) should be reviewed, as it may be a dependent variable in the experimental system. Similarly, the experimental procedures and conditions should be reviewed. In Figure 4B, KLF2 knockdown using siRNA yields similar knockdown efficiency between three separate experiments (Experiment 1, 2, and 3). We used three distinct biological samples for these experiments, using two simultaneously running flow chambers with shear stress at 1 Pa for each experiment.
Figure 1: Agarose gel image of the linearization of exogenous luciferase plasmid. Supercoiled and 1 kb+ DNA ladders are used as markers to determine both uncut and cut luciferase plasmid sizes in kilobases (kb). Please click here to view a larger version of this figure.
Figure 2: Schematic overview of multiple flow circuit assemblies within a dedicated environment (the BEACH). The flow rates in both flow circuits are easily monitored in real-time, without disturbing the environment, and both circuits can run simultaneously. Please click here to view a larger version of this figure.
Figure 3: Schematic overview of a single flow circuit assembly. The tubing sizes and Luers used are indicated in this figure. Ensure that the flow meter is oriented in the direction of the flow and placed upstream of the flow chamber. Please click here to view a larger version of this figure.
Figure 4: Representative results from KLF2 loss-of-function experiments in human endothelial cells exposed to shear stress (1 Pa) for 24 h. (A) This panel shows the quantification of exogenous luciferase RNA in three separate flow experiments. Luciferase efficiencies can be similar between samples of an experiment (Experiment 1) or show some variability (Experiments 2 and 3). Luciferase (absolute copies) is the copy number of luciferase RNA detected by reverse-transcriptase quantitative PCR (RT-qPCR) by absolute quantitation using a standard curve. Luciferase efficiency is the experimental luciferase copies divided by the theoretical luciferase copies for each sample multiplied by 100 (see step 8 of the protocol). Relative luciferase efficiency is the luciferase efficiency of each sample divided by the reference condition (Flow + Ctlsi) within each experiment. (B) These panels show the normalization of gene expression in a set of sample shear stress experiments using both endogenous and exogenous reference genes. The results are from reverse-transcriptase quantitative PCR (RT-qPCR). Knockdown of KLF2 mRNA expression is shown in the presence of laminar flow with shear stress of 1 Pa for 24 h. FC = fold change; CycA = cyclophilin A, used as an endogenous reference RNA; Luc = luciferase, used as an exogenous reference RNA. The data adapted from Man et al.5. Please click here to view a larger version of this figure.
Chamber Height (microns; µm) |
Chamber Width (centimeters; cm) |
Flow Rate (mL/min) for 1 Pa shear stress | Viscosity (centipoise; cP) |
303.80 | 1.90 | 19.48 | 0.90 |
326.10 | 1.90 | 22.45 | 0.90 |
344.84 | 1.90 | 25.10 | 0.90 |
319.06 | 1.90 | 21.49 | 0.90 |
Table 1: Chamber heights and examples of flow rates for various flow chambers to achieve shear stress of 1 Pa.
J cloths |
Tweezers |
Reservoir Bottle and Cap |
Dampener and Cap |
Flow Loop |
1/16" Male Luer x 3 |
1/16" Female Luer x 3 |
1/8" Male Luer x 2 |
1/8" Female Luer x 4 |
3/16" Male Adaptor x 2 |
14 L/S Hard Tube (2 inches) x 1 |
14 L/S Soft Tube (5 inches) x 2 |
16 L/S Soft Tube (3 inches) x 2 |
25 L/S Soft Tube (3 inches) x 2 |
13 L/S Hard Tube (10 inches) x 1 |
Flow Chamber |
1/8" Male Luer x 2 |
1/8" Female Luer x 2 |
16 L/S Soft Tube (3 inches) x 3 |
Top and Bottom Plates |
Screws |
Table 2: Parts to be autoclaved in step 6.2 of the protocol.
Shear stress is a physiologic condition that modulates endothelial function, in part, by affecting steady-state gene expression2,5. Models of gene regulation in various shear stress conditions will contribute to a greater understanding of endothelial function. This pragmatic workflow includes a flow circuit using a parallel-plate flow chamber adapted from Lane et al.9 and represents laminar, non-pulsatile flow. The overall set-up was designed to facilitate experiments that require multiple flow chambers and minimize experimental variability in this setting.
The flow circuit assembly is a major component of this workflow and is adapted from Lane et al.9. Several adaptations of this assembly and protocol were made to reflect differences in the experimental systems. A large heated unit, the BEACH, is an adaptation that facilitates the simultaneous operation and monitoring of several flow circuits within the same environment. This system has been used successfully for the application of shear stress to endothelial cells for various time periods, from 1 h to 7 days, and at several levels of shear stress (e.g., 1.0, 1.5, and 2.0 Pa). This system was also used for gene knockdown studies to assess the function of a flow-responsive endothelial gene in the setting of shear stress (Figure 4)5. There is considerable variability between pumps and pump heads, which may also change over time due to normal wear and tear. To account for these differences, we use a flow sensor situated proximal to the flow chamber to continuously monitor flow rates. Various sizes of tubing and Luers are used to ensure a tight fit for each component and to prevent any leakage during experiments. We counted cells and seeded approximately 1,000,000 cells per glass slide, dropwise, and then let the cells incubate at 37 °C for 15 min to increase adherence efficiency. Compared to endothelial progenitor cells, which can be seeded for a short time prior to the application of shear stress9, we seed human endothelial cells for at least 24 h prior to the application of shear stress. Shorter durations can lead to the dislodging of cells during flow, or a discontinuous layer of endothelial cells, even with the appropriate seeding density. We emphasize the inspection of the slides for a confluent monolayer of endothelial cells both prior to and after the application of shear stress. We find that slides coated with fibronectin, a natural extracellular matrix component18, maintain the endothelial monolayer more consistently compared to sides coated with gelatin (denatured fibrillar type I collagen). Finally, the flow dampeners in this protocol are optimized to use 30 mL of media, compared to 190 mL of media for other manufacturers.
Several steps in the flow circuit assembly require additional care. An even monolayer of endothelial cells should be established prior to the application of shear stress. It is important to seed cells onto the glass slide dropwise to increase the number of cells that adhere to the slide and, thus, the overall slide coverage. The wait time between cell seeding and adding additional media to the slide generally improves seeding efficiency as it provides sufficient time for cells to adhere to the slide rather than be washed away into the multi-slide tray. Inspect cells visually before, during, and after the application of shear stress. A microscope can be placed in the BEACH for this purpose. The flow sensor must be attached in the correct orientation and the target flow rate should be checked for each individual flow chamber. The flow loop system should be perfused without the chamber to ensure no media leakage or other problems, such as pressure build-up, to avoid perturbing the cells during the actual experiments. Inspect for and eliminate bubbles in the system. While testing the flow loop system, ensure the stopcocks are all open before turning on the peristaltic pump to allow uninterrupted, unidirectional flow. Ensure that all stopcocks are closed prior to attaching the flow chamber to the loop and fully opened prior to restarting the pump.
We find that the addition of an exogenous reference gene is helpful in a variety of scenarios. Endothelial cell media often contains heparin, which is an inhibitor of PCR17. While some RNA extraction protocols incorporate steps to remove heparin, trace amounts can cause differences in PCR efficiencies between samples. Potent treatments may also preclude the identification of an endogenous reference gene that is quantitatively stable. Our lab has created an efficient protocol to synthesize 5'-capped and poly-A-tailed luciferase RNA for use as an exogenous reference RNA. This strategy has proved to be a cost-effective approach compared to purchasing a commercially available RNA. During the preparation of exogenous reference RNA, it is important to aliquot RNA into single-use aliquots to avoid multiple freeze-thaw cycles. Thorough mixing and accurate pipetting are critical to maintaining inter-aliquot consistency. Typical experiments show a luciferase efficiency in the range of 5% ± 2.5% but can range from 1% – 10%. It is prudent to correct RT-qPCR results for both the exogenous reference RNA efficiency and an endogenous reference (housekeeping) gene.
For the experiments we conduct, firefly luciferase sequences are used as a non-mammalian spike-in reference RNA in mammalian models. In experiments where firefly luciferase is expressed in cells, this would not be an appropriate reference gene. Other species-specific reference genes can be used, including the Caenorhabditis elegans miRNA cel-miR-3919 and ribulose bisphosphate carboxylase plant RNA20.
This flow circuit models a 2-D monolayer of endothelial cells grown on tissue culture plastic or glass, which is quite stiff. Matrix stiffness can influence the endothelial response to fluid shear stress21. This model system uses a relatively high oxygen concentration more similar to arterial than venous oxygen concentrations. This model closely resembles straight segments of larger vessels in a closed cardiovascular system and provides a relatively homogenous environment for endothelial cells on the slide. Other specific conditions in 3-D structures, such as bifurcations or curvatures of vessels, are not represented with this model. Other systems can model other flow patterns, including those in curved regions of the vasculature, but yield fewer cells than the system described in this protocol. Similarly, other assemblies may be more appropriate if > 1 x 106 cells are required, or if a single-cell analysis is required. Our current application models non-pulsatile laminar flow. Yet, this model can be used to generate other waveforms, including pulsatile or oscillatory waveforms, with consistency, as the flow rates are monitored continuously.
Overall, this pragmatic workflow provides a system for the simultaneous application of shear stress to multiple flow chambers with monitored flow rates. Materials and procedures throughout this workflow are designed to minimize experimental variability between samples and conditions. This workflow has been successfully used for RNAi experiments in the setting of laminar flow and can be also used for any experiments requiring multiple conditions with laminar shear stress, or multiple laminar shear stress magnitudes and/or time points, including alternative waveforms.
The authors have nothing to disclose.
This work was supported by CIHR MOP 142307 to P.A.M. H.S.J.M. is a recipient of a Canadian Institutes of Health Research Training Program in Regenerative Medicine Fellowship. H.S.J.M., A.N.S., K.H.K., and M.K.D. are recipients of the Queen Elizabeth II Graduate Scholarships in the Science and Technology.
0.05% Trypsin-EDTA | gibco | 25300-062 | |
10 mL Syringe | BD | 302995 | |
10 mm2 Culture Dish | Sarstedt | 83.3902 | |
30 mL Syringe | BD | 302832 | |
4-Way Stopcocks | Discofix | D500 | |
Aluminum foil | |||
BEACH | Darwin Chambers Company | MN: HO85, SN: 4947549 | |
Cell Scrapers | |||
CO2 Meter | BioSphenix, Ltd. | MN: P120, SN: 0342 | |
CO2 Sensor | BioSphenix, Ltd. | MN: C700, SN: 52852 | |
Distilled water | gibco | 15230-170 | |
Dulbecco's phosphate-buffered saline (DPBS) -/- | gibco | 14190-144 | |
Endothelial Cell Growth Medium 2 | Promo Cell | C-22011 | |
Endothelial Cell Growth Medium 2 Supplement Mix | Promo Cell | C-39216 | |
Fibronectin (pure) | Sigma-Aldrich | 11051407001 | |
Filter (0.20 um) | Sarstedt | 83.1826.001 | |
Flow Dampener and Cap | U of T glass blowing shop | ||
Flow Meter: 400 Series Console | Transonic Scisense Inc. | T402 | |
Flow Meter: 400 Series Tubing | Transonic Scisense Inc. | TS410 | |
Flow Reservoir and Cap | U of T glass blowing shop | ||
Flow Sensor | Transonic Scisense Inc. | ME4PXL | |
Isotemp 737F Oven | Fisher Scientific | FI-737F | |
J cloth | J cloth | ||
Microscope Slide (25 x 75 x 1 mm) | Fisherfinest | 12-544-4 | |
Paper sterilization pouch | Cardinal Health | 92713 | |
Pump (Masterflex L/S Economy Drive) | Cole-Parmer | 7554-90 | |
Pump Head (Masterflex L/S Easy Load) | Cole-Parmer | 7518-00 | |
Rectangular 4 Well Dish | Thermo Scientific | 267061 | |
Tweezers | |||
Name | Company | Catalog Number | Yorumlar |
Tubing | |||
Masterflex C-Flex L/S 25 Soft Tubing | Cole-Parmer | 06424-25 | |
Masterflex C-Flex L/S 14 Soft Tubing | Cole-Parmer | 06424-14 | |
Masterflex C-Flex L/S 16 Soft Tubing | Cole-Parmer | 06424-16 | |
Masterflex PharMed BPT L/S 13 Hard Tubing | Cole-Parmer | 06508-13 | |
Masterflex PharMed BPT L/S 14 Hard Tubing | Cole-Parmer | 06508-14 | |
Name | Company | Catalog Number | Yorumlar |
Luer | |||
3/16" Male Luer | Cole-Parmer | 45518-08 | For #25 tubing |
1/8" Male Luer | Cole-Parmer | 30800-24 | For #16 tubing |
1/8" Female Luer | Cole-Parmer | 30800-08 | For #16 tubing |
1/16" Male Luer | Cole-Parmer | 45518-00 | For #14 tubing |
1/16" Female Luer | Cole-Parmer | 45508-00 | For #14 tubing |
Name | Company | Catalog Number | Yorumlar |
Knockdown reagents | |||
Oligofectamine Reagent | Invitrogen | 12252-011 | |
Opti-MEM I Reduced Serum Medium | gibco | 31985-070 | |
Name | Company | Catalog Number | Yorumlar |
In vitro transcription | |||
Generuler 1kb+ DNA ladder | Thermo Scientific | SM1331 | |
MEGAclear Kit | Ambion | AM1908 | |
mMESSSEGE mMACHINE SP6 Transcription Kit | Ambion | AM1340 | |
pSP-luc+ | Promega | E4471 | |
Supercoiled DNA Ladder | New England BioLabs Inc. | N0472S | |
UltraPure Agarose | Invitrogen | 16500-500 | |
UltraPure Ethidium Bromide | Invitrogen | 15585011 | |
XhoI Restriction Enzyme | New England BioLabs Inc. | R0146S | |
Name | Company | Catalog Number | Yorumlar |
RNA extraction | |||
Beta-mercaptoethanol | Sigma | M3148-100mL | |
RNeasy Mini Kit | Qiagen | 74104 |