Biology Reference
In-Depth Information
the ratio 1,000
L is the dilution factor to calculate the
copy number per milliliter, considering that a volume of 5
μ
L/5
μ
μ
L
of template was added to the reaction.
3.4 Infections
of Cells with HD
Adenoviral Vectors
1. Huh7 are maintained in 100 mm culture dishes. 1 × 10
6
cells
are seeded in 60 mm dishes and after 48 h, cells are detached
for counting. Cells are incubated with HD adenovectors at a
multiplicity of infection (MOI) of 1,000 vector genomes per
cell, 1 h, 30 min at 37 °C in D-MEM supplemented with 5 %
FBS, rocking the plate every 20 min. Then, cells are washed
twice with PBS, and medium is replaced with fresh complete
medium until RNA extraction at the different time points.
2. hmNPCs are seeded in T-25 cm
2
fl asks and, when 90-100 %
confl uent, are incubated 5 days with differentiation medium.
Then, cells are incubated with adenoviral vectors at an MOI of
1,000 vector genomes per cell, 2 h in differentiation medium
at 37 °C, 5 % CO
2
and 3 % O
2
, shaking the cells every 20 min.
Afterwards, cells are washed twice with PBS and the RNA is
collected at the different time points.
1. Microarray analysis requires a highly purifi ed RNA generally
achieved by using columns after the cell lysis and a digestion of
contaminating DNA with DNaseI.
2. The commercial Qiagen RNeasy mini kit for RNA cleanup can
be used both on plated cells and after TRIzol extraction and
includes the step for on-fi lter DNase digestion to remove
genomic DNA.
3. To effectively purify smaller RNA transcripts such as <200 bp
appropriate protocols and kits with effi cient enrichment of
microRNA should be taken into consideration.
4. At desired time points the medium is removed and 1 mL of
Qiagen RNeasy mini kit RPL lysis buffer is added to the cells
in T-25 cm
2
fl ask mixing well.
5. Using the cell scraper, cells are collected with lysis buffer on
one side of the fl ask. Holding the fl ask inclined, the lysis solu-
tion is completely aspired and the lysate is transferred to 2 mL
tubes.
6. The lysate is further homogenized by vortexing 1 min. One
volume of 70 % ethanol is added to the lysate and vortexed
thoroughly. 700
3.5 RNA Extraction
for Microarray
Hybridization
L of the sample are applied to the column
placed in a 2 mL collection tube and centrifuged for 15 s at
≥
μ
8,000 ×
g
at room temperature (RT). The fl ow-through is
discarded. This step is repeated by transferring the rest of sam-
ple to the column.
7. Add 350
L of Qiagen RNeasy mini kit RW1 wash buffer to
the column and centrifuge for 15 s at
μ
≥
8,000 ×
g
at RT. The
fl ow-through is discarded.
