Osteocyte Differentiation: ALP Staining and RT-PCR

Question 1

Types of cells mesenchymal stem cells forms

Answer 1

chondrocytes, cardiomyocytes, osteoblasts, adipocytes, neurons, myocytes

Question 2

Key proteins of osteogenesis

Answer 2

alkaline phosphatase, osteocalcin, runx2

Question 3

Bone morphogenic protein- 2

Answer 3

member of the TGF-beta superfamily
26 kD homodimeric protein- joined by sulfide bond
composed of 2 identical 115 aa chains
100% conservation between humans, mouse, and rat proteins

Question 4

Runx2

Answer 4

master switch for cells to go to osteocytes

Question 5

Dlx5, Dlx3, Msx2

Answer 5

transcription factors that sit on regulatory elements that prevents Runx2 from being transcribed

Question 6

Runx2

Answer 6

master switch for cells to go to osteocytes

concentration depends on the cell type

Question 7

Dlx5, Dlx3, Msx2

Answer 7

transcription factors that sit on regulatory elements that prevents Runx2 from being transcribed
necessary to keep in differentiation

Question 8

Models of mesenchymal stem cell differentiation

Answer 8

divergence, dedifferentiation, trans-differentiation (true model)

Question 9

Results of expression of MyoD and ALP during differentiation

Answer 9

expression of MyoD was down regulated after the induction of ALP
the down regulation of MyoD is not necessary to begin osteogenic differentiation

Question 10

Steps for isolating RNA from cells

Answer 10

cells get lysed and homogenized –> add ethanol –> bind total nucleic acids (including genomic DNA and RNA) –> was 3 times –> elute

Question 11

Cell lysis

Answer 11

solution 1 (RLT buffer) which contains guanidine thiocyanate and BMA

Question 12

Homogenization

Answer 12

tuberculin syringe to shear high-molecular weight genomic DNA and cellular components

Question 13

Ethanol

Answer 13

added to lysis buffer to optimize binding of RNA to membrane

Question 14

Solution 2 (RW1)

Answer 14

washes membrane

Question 15

DNaseI solution

Answer 15

chews up contaminating DNA

Question 16

Solution 3 (RPE)

Answer 16

washes membrane

Question 17

Requirements of first strand cDNA synthesis

Answer 17

oligo(dT)- primer
dNTPs
0.1 M DTT
reverse transcriptase

Question 18

Primer

Answer 18

binds to polyA tail

Question 19

Primer

Answer 19

binds to polyA tail

Question 20

0.1 M DTT

Answer 20

stabilizes reverse transcriptase activity

Question 21

Reverse transcriptase

Answer 21

generates complementary DNA (cDNA) from RNA templates

Question 22

Requirements of RT-PCR

Answer 22

taq polymerase
PCR buffer
MgCl2
dNTPs
Primers

Question 23

Taq polymerase

Answer 23

heat-stable DNA polymerase

Question 24

PCR buffer

Answer 24

provides suitable chemical environment

Question 25

MgCL2

Answer 25

required cofactor for polymerase activity

Question 26

dNTPs

Answer 26

nucleotides containing triphosphate groups