Application Note 40

Gene Expression Profiling with qPCR
Applicable for Cells Cultivated in Channel-Slides
Gene expression profiling provides information about the transcriptome of a livingcell. The DNA is transcribed into mRNA, when the specific gene is active. This canbe influenced by stimuli like chemical compounds, internal signals of the cell ormechanical stress.
This Application Note describes the preparation of cell lysates, directly in the ibidi
channel-µ-Slides, and the subsequent quantification of mRNA in HUVEC. The mRNA
is isolated from cells and analyzed afterwards with quantitative PCR (qPCR). In thisexample, the detection of Claudin-5 in HUVEC is presented. The protocol consists offour main steps as seen in Figure 1.

Figure 1: Four main steps for gene expression profiling.

Table of Contents:
0. Material ................................................................................................................ 2
1. Cell Cultivation ..................................................................................................... 3
2. RNA Extraction .................................................................................................... 3
3. Reverse Transcription .......................................................................................... 6
4. Quantitative PCR ................................................................................................. 6_____________________________________________________________________________________________________Application Note 40© ibidi GmbH, Version 1.1, March 8th, 2018Page 1 of 7Application Note 40

0. Material
Cell Culture

HUVEC (ca. 2 x 105 cells)µ-Slide I 0.4 Luer (one slide)
Optional: ibidi Pump System for flow conditioning of cells
Appropriate cell culture medium and reagents for cell splitting

Cell Lysis and Homogenization


Lysis Buffer (RLT Buffer from the Kit)
PBS
20-gauge needle and syringe

RNA Extraction

RNeasy Micro Kit (Qiagen)Ethanol (70% and 96%)
Ethanol 80% with RNAse-free water

Reverse TranscriptionQuantiTect Reverse Transcription Kit (Qiagen)Optional: gene-specific primer

Quantitative PCR
 Light Cycler 480 SYBR Green (Roche) Gene-specific primer and GAPDH primer
Equipment


CentrifugeVortex
Sterile, RNAse-free pipet tips
Reaction cups (2 ml / 200 µl)
Microplate Spectrometer
Heating block_____________________________________________________________________________________________________
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1. Cell Cultivation
Cultivate the cells inside a channel slide, and then treat them to meet your special
experimental needs.
In our example, we show the gene expression profiling with flow-cultivated HUVEC inµ-Slides I 0.4 Luer. The cells were seeded in the Slides and connected to the flow
conditions after the time period needed for cell attachment. At several time points,
samples were disconnected and prepared following the steps of this protocol, todetermine the time response of the gene expression. As gene of interest we chose atight junction protein (Claudin-5), which is only present in endothelial cells, and whichis strongly regulated by flow conditions.
The setup of an experiment to cultivate HUVEC under shear stress conditions isdescribed in detail in Application Note 13.

Figure 2: Flow cultivated HUVEC at 30 dyn/cm² after a period of one day. The arrow indicates thedirection of flow.

2. RNA Extraction
In the first step, RNA is extracted from the cells of interest. The total mRNA
represents the spectrum of genes which is active at the actual time point.
The RNeasy Micro Kit from Qiagen was used to isolate total RNA from flow-cultivated
HUVEC. This kit is easy to handle and enables the selective binding of RNA to asilica-based membrane.
Differing from the manufacturer’s instructions, some improvements to the protocol
have been made to get the best results, related to the quality and quantity of RNA.
For further information about the kit, please read the RNeasy Micro Kit Handbook ([1]Qiagen).
Important Note!
This Application Note is optimized for
the RNA extraction of HUVEC.

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2.1. Cell Lysis and Homogenization
It is important to perform all of the following steps at room temperature (15–25°C)
and to work as fast as possible. Homogenized cell lysates can be stored at –70°C.
For further instructions, please read the RNeasy Micro Kit Handbook [1] (Qiagen).

1.
2.
3.
4.
5.

6.

Aspirate the cell culture medium from the channel slide.
Wash the cells four to five times with 120 µl of PBS.
Remove the PBS from the channel.
Add 100 µl of RLT Buffer to the channel and plug the syringe into one Lueradapter (Figure 3A).
Shear the cells out of the channel by moving the plunger quickly up and down.
Be careful not to squeeze out the cell lysate at the empty Luer adapter of theslide.
Repeat Steps 4 and 5 one time.
AB

0.4

Figure 3: A) µ-Slide I Luer filled by the pipette. Lyse cell layer in channel slide by moving theplunger up and down. B) Homogenize the cells with syringe and needle by moving the plunger up anddown.

7.
8.
9.

Mix the disrupted cells with 150 µl of RLT Buffer, to achieve a total volume of350 µl.
Slip a needle onto a new syringe and pass the lysate through the needle at least
10 times (figure 3B).
Vortex the solution for 30 seconds to homogenize the lysate completely.

2.2. Extraction (According to the Qiagen protocol [1])Bind total RNA1.)

Add 350 µl of 70% ethanol to the lysate and mix by pipetting.

2.)

Transfer the sample to an RNeasy MinElute spin column. Close the lid andcentrifuge for 15 seconds at ≥8000 x g. Discard the flow-through.

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Digest DNA3.)

Add 350 µl of RW1 Buffer to the column. Close the lid and centrifuge for
15 seconds at ≥8000 x g to wash the column. Discard the flow-through.

4.) Add 10 µl of DNase I stock solution to 70 µl RDD Buffer and mix gently.
5.)

Add the DNase I incubation mix directly to the column membrane and place itfor 15 minutes at room temperature.

6.)

Add 350 µl of RW1 Buffer to the column. Close the lid and centrifuge for
15 seconds at ≥8000 x g to wash the column. Discard the flow-through.

Wash
7.)

Place the spin column in a new 2 ml collection tube.

8.)

Add 500 µl of RPE Buffer, then close the lid and centrifuge for 15 seconds at≥8000 x g to wash the membrane. Discard the flow-through.

9.)

Repeat Step 8.

10.) Add 500 µl of 80% ethanol to the column, close the lid and centrifuge for 2
minutes at ≥8000 x g. Discard the flow-through and the collection tube.
Elute11.) Place the spin column in a new 2 ml collection tube. Open the lid andcentrifuge at full speed for 5 minutes. Discard the flow-through and collectiontube.
12.) Place the RNeasy MinElute spin column in a new 1.5 ml collection tube. Add
14 µl of RNAse-free water to the membrane and incubate for 3 minutes. Closethe lid and centrifuge for 1 minute at full speed to elute the RNA.
13.) Storage is possible at –20°C in RNAse-free water for several weeks.

The concentration of RNA can be determined by measuring the absorbance at
260 nm directly from the isolated sample. The quotient of the absorbance at 260 nm
and 280 nm provides an estimated purity of RNA.
A maximum yield of 1.5 µg RNA is expectedfor this amount and type of cells.

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3. Reverse Transcription
In the reverse transcription step, the RNA is transferred into complementary DNA(cDNA), which is much more stable for the use in the subsequent PCR.
We used the QuantiTect Reverse Transcription Kit (Qiagen) according to themanufacturer’s instructions. For further information about this kit please read theQuantiTect Reverse Transcription Kit Handbook.

4. Quantitative PCR
The quantitative PCR shows the increment of targeted DNA in real time. A DNAbinding dye is used, which only binds to double stranded DNA. This binding induces
fluorescence which is then detected in real time and thus can be used as aquantitative marker for the amount of DNA.
However, this method is not capable to detect absolute quantification of the sample.
The targeted DNA is compared to a housekeeping gene, which is not affected by theexperimental conditions. Like this, a relative quantification of the various experimental
conditions can be determined. Sample data are shown in the graph below.
In our protocol, we used the Light Cycler® 480 SYBR Green I Master mix to performthe real-time PCR with the Light Cycler® 480 instrument. For further information
please read the Light Cycler handbook ([2] Roche).
In our example, GAPDH is used as a reference gene, because it is not regulated
under flow conditions [3]. The gene of interest is a tight junction protein, which isstrongly regulated by flow conditions. The primers are given in the table below.
Table 1: Primer sequence for Claudin-5 and the reference gene GAPDH.

NamehGAPDH_forward
hGAPDH_reverse
Claudin-5_forward
Claudin-5_reverse

Sequence
ATG GGG AAG GTG AAG GTC G
GGG GTC ATT GAT GGC AAC AAT A
CGG GTG TCA GAC TGA GGA TT
CCT GCC GAT GGA GTA AAG AC

The following heating steps were used to amplificate the cDNA template (preliminarydata, the settings might still be optimized).
Table 1: Light cycler® program.

Steps
Pre-Incubation:
Amplification:

Temperature
95°C
95°C
60°C
72°C

Time5 min
10 s
10 s
10 s

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5. Example Result
As an example we show here preliminary results of the Claudin–5 relative gene
expression in HUVEC at several time points during cultivation under flow conditions(internal data).

Figure 4: Relative Gene expression of thetight junction protein Claudin-5

The gene expression of the cultured cells before applying the flow (0 h) is assumed
as 100%. After three hours of flow the gene expression goes clearly down andrecovers after 24 hours.

Sources:[1] Qiagen, RNeasy Micro Handbook, Second Edition, December 2007.
[2] Roche Diagnostics GmbH, Light Cycler 480 Instrument, Operator’s Manual,
Version 1.5, 2008
[3] R. D. Barber, D. W. Harmer, R. A. Coleman, B. J. Clark, GAPDH as ahousekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 humantissues. Physiological genomics 2005, 21 (3), S. 389–395.

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