PCR i inne techniki

molekularne

Krzysztof Giannopoulos

Zastosowanie metod

wykrywania antygenu w

zależności od etapu

syntezy białka

•

DNA

•

RNA

•

Polipeptyd

•

Modyfikacja
wewnątrzkomórkow
a

•

Wydzielanie

•

Modyfikacja
zewnątrzkomórkowa

• PCR
• RT PCR
• Western blotting
• cytometria

wewnątrzkomórko
wa

• cytometria

przepływowa

• ELISA

Techniki analiz

genetycznych

• Southern blot (SB) analysis
• Polymerase chain reaction (PCR), and

real-time quantitative PCR (RQ-PCR)

• konwencjonalna cytogenetyka
• Fluorescence in situ hybridisation (FISH)
• comparative genomic hybridisation (CGH)
• gene expressions profiling (GEP)

TRI Reagent® is a single, homogenous solution for the isolation of total RNA. This
phenol-based reagent contains a unique combination of denaturants and RNase
inhibitors and is used in a convenient, single-step disruption/separation procedure.
The tissue or cell sample is homogenized or disrupted in the TRI Reagent, chloroform
is mixed with the lysate, and the mixture is separated into three phases by
centrifugation. The RNA is then precipitated from the aqueous phase with
isopropanol. The entire procedure can be completed in no more than one hour to
produce high yields of intact RNA for use in Northerns, nuclease protection assays, RT-
PCR and in vitro translation. TRI Reagent is especially effective for purifying RNA from
microorganisms.
Description: TRI Reagent is an improved version of the single-step total RNA isolation
reagent developed by Chomczynski (1). The RNA isolation method based on this
reagent is widely recognized and proven for RNA applications and is supported by a
substantial publication list (2,3,4,5). It is ideal for quick, economical, and efficient
isolation of total RNA or the simultaneous isolation of RNA, DNA and proteins from
samples of human, animal, plant, yeast, bacterial and viral origin.

References
1. Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156
(1987).

2. Chomczynski, P., A reagent for the single-step simultaneous isolation of RNA, DNA
and proteins from cell and tissue samples. BioTechniques 15: 532-537 (1993).

3. Mackey, K. and Chomczynski, P., Long-term stability of RNA isolation reagents. J. NIH
Res. 8: 72 (1996).

4. Wilfinger, W., et al., Effect of pH and ionic strength on the spectrophotometric
assessment of nucleic acid purity.BioTechniques 22: 474-481(1997).

5. Chomczynski, P. and Mackey, K., Modification of the Tri Reagent. procedure for
isolation of RNA from polysaccharide- and proteoglycan-rich sources.BioTechniques
19: 924-945(1995).

aqueous phase (RNA)
interphase
Phenol-chlorophorm (proteine + DNA)

Jakość izolowanego RNA

Reakcja odrwotnej transkrypcji

RT (reverse transcription)

Rodzaje RT-PCR

• RT-PCR (reverse transcriptase polimeraze

chain reaction) reakcja lancuchowej

polimerazy z odwrotna transkrypcja

• qRT-PCR (quantitative RT-PCR) – ilosciowy

RT-PCR

• rtRT-PCR („real time“ RT-PCR) – ilosciowy

RT-PCR

• Competitive RT-PCR
• RT-PCR „in situ“
• RT-PCR muliplex

Quantify relative opsin gene

expression

• Make total RNA from retinas of single adult fish

–

Trizol

–

Quantify RNA by A260/A280

• Reverse transcribe 1 ug of total RNA using T

17

primer and

Superscript II

• Make master mix of PCR reagents for each fish

–

15 ul 2x Taqman ready made PCR mix

–

1 ul

RT reaction

–

5 ul water

– 21 ul

• Make primer / probe master mix for each gene of interest

–

3 ul forward primer (3 uM)

–

3 ul reverse primer (3 uM)

–

3 ul Taqman probe (2 uM)

–

30 ul total

Run real time thermal cycle

50 C

5 min

95 C

10 min

20 s

55 C

30 s

65 C

1 min

40 cycles

Activates
UNG
enzyme

Activates
Amplitaq
Gold

Screening BAC library for

red opsin gene

0

2

4

6

8

10

12

14

16

0

10

20

30

40

PCR cycle

R

n

SP5
SP6
SP7
SP8
SP9
SP10
Genomic

Dissociation curves: BAC

library screen for red opsin

0

1

2

3

4

5

6

65

70

75

80

85

90

Temperature

D

e

ri

v

a

ti

v

e

o

f

R

n

SP5
SP6
SP7
SP8
SP9
SP10
Genomic

Competitive RT-PCR

Measurement of two RNA samples.
The lower bands are the standard bands, the upper bands are the
transcript bands. Lane 1-4: sample 3; lane 5-8: sample 4; lane 1+5: 250
pg standard; lane 2+6: 100 pg standard; lane 3+7: 50 pg standard; lane
4+8: 25 pg standard. The rectangles represent objects set with the
ImageQuant software.

Znacznik
wielkosci

Molecular

Weight Marker

VI

Znaczniki używane do analizy

cyklu komórkowego w

cytometrze przepływowym:

•UV excited: Hoechst 33342, Hoechst 33258, DAPI

•457nm excited: Mithramycin

•488nm excited: Propidium Iodide,

7Aminoactinomycin D, SYTOX Green, DRAQ5

•633nm excited: TO-PRO-3 Iodide

PROCEDURE:
•

Isolate cells:

•

1. For adherent cells, remove with PBS/EDTA and/or trypsin solution. For cells in

suspension, harvest by

•

centrifugation. Centrifuge cells at 1200 rpm at 4°C for 5 minutes, decant supernatant

and gently re-suspend

•

the cells in PBS.

•

2. Count the cells by hemocytometer.

•

3. Wash cells one time by putting 1X105 cells per tube, adding 1 ml of PBS and

centrifuging at 1200 rpm at

•

4°C. Re-suspend pelleted cells in 0.3 ml of PBS buffer.

•

Fixing the cells:

•

1. To fix the cells, gently add 0.7 ml cold ethanol (70%) dropwise to tube containing

0.3 ml of cell suspension in PBS while vortexing gently.

•

2. Leave on ice for 1 hour (or up to a few days at 4°C).

•

3. Centrifuge cells as above, wash 1 time with cold PBS and re-centrifuge.

•

4. Re-suspend cell pellet in 0.25 ml of PBS, add 5 µl of 10 mg/ml Rnase A (the final

concentration being

•

0.2-0.5 mg/ml).

•

5. Incubate at 37°C for 1 hour.

•

6. Add 10 µl of 1 mg/ml PI solution (the final concentration being 10µg/ml). Keep in

the dark and at 4°C until

•

analysis.

•

7. Analyze on FACS by reading on cytometer at 488 nm.

•

Propidium iodide stain (PI): Make a 1 mg/ml solution of PI in deionized

water.