Gene copy number and transcript analysis

Preparation of genomic DNA and cDNA

Genomic DNA (gDNA) was prepared from stable recombinant CHO cell lines. 2×10

cells were harvested and washed with PBS. gDNA was isolated and purified using

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

7

the QIAamp® DNA Blood Mini kit (Qiagen) according to the manufacturer’s

instructions.

Total RNA was isolated from 2×10 cells using Ambion Tri Reagent Solution (Life

Technologies, CA) according to the manual. DNA contaminations were removed from

extracted RNA by digestion with DNase (Qiagen, Netherlands) in the presence of

RNase inhibitor (Life Technologies, CA). Purified RNA was stored in RNAse-free

water at −20°C. Total RNA was reverse transcribed using M-MLV reverse

transcriptase kit (Promega) according to manufacturer’s instructions. Purity and

concentration of the gDNA and cDNA was determined with the ND-1000

Spectrophotometer (Nano-Drop®).

qPCR

qPCR was performed with the MiniOpticon system (BioRad) utilizing the Taqman

method. Primers were designed to amplify a 100 bp fragment in the Fc part of the two

scFc antibody derivates. The β-actin gene was amplified as internal control and to

normalize the data. Amplification was either performed with gDNA or cDNA by

denaturation at 95°C for 5 minutes followed by 40 cycles at 95°C for 15 seconds and

55°C for 1 minute. Fluorescence signal and cq-values were calculated by the CFX

manager software (BioRad). For relative quantification the 2 method (Livak and

Schmittgen 2001) was used. To obtain statistical significant data three biological

samples of each cell clone were analyzed in triplicates. Statistical analysis was

performed with the SPSS 18 software package (IBM).

You can also use the RNA W-curve to visualize the RNA (see slideshare/lembark)

You might want to consider both qPCR and western blot. Do qPCR for all the genes you are interested in. Do western blots for those you are most interested in or that change, since antibodies for westerns are more expensive than primers. We often screen with qPCR and save westerns for validation. But in the end, determining the expression level and biochemical activity of the protein directly is best.

I suggest you do both for they do differ in expression levels during treatments.

Amanda, would like to use millipore multiplex but unfortunately we don't have such resources here. My main concern is, that do mRNA level of eIFs really reflect changes in their levels- if somebody has looked at it? For instance the I usually draw conclusions that high mRNA = high protein (which i can later verify) and viceversa. However in certain cases, the mRNA levels are low, which means transcription has stopped however the protein is sitll present in high levels. I am wondering does low mRNA possibly mean that its low because a lot of it is being processed to be made into protein. Surely transcription factors for general cell biology are required all the time, and if a stimulus makes their mRNA go low- what does it mean?

I'll just add that a lot of recent high-throughput analyses have been done on global RNA half-lives. I would do a literature search. I'm sure data for any of your genes is freely available and probably for a related cell type. The gold standard is still protein levels and, if possible, enzymatic activity of the specific proteins. Transcript levels can be deceiving and you really care about how much functional enzyme is present.

Hi Mustafa,

There are a lot of good antibodies against a range of eIF's which work well in whole cell and tissue lysates so measureing the protein is pretty easy and WILL be much more representative of the activity of what you are interested in. Additionally there are now sepharose beads conjugated to an mRNA 5'Cap whcih you can use to pull out active cap binding complexes. Again this is fairly easy and will be a lot more representative of the cap binding capacity and availability of eIF4E for instance. There are also a number of phospho-specific antibodies which are good indicators of eIF function. The eIF2alpha phospho antibodies are a really good readout of the ability of eIF2alpha to drive initiation - if it is phosphoryalted initiation stops.

Let me know if you need any pointers to good antibodies. I would reccomend a translation initiation regulation kit from NewEnglandBiolabs. Its about £300 and you get small aiquots of a bunch of antibodies against eIFs and others.

All the best,

Lee.

Hi,

many of the translation factors are regulated post translational and therefore it is important to assess them at protein level (immunoblotting) with antibodies sensitive to the modification, anti phospho etc. Furthermore, it may be important to assess their activity by measuring targets levels- depends on the specific translation factors you are interested in.